Evidence for gene essentiality in Leishmania using CRISPR

Wen-Wei Zhang; Greg Matlashewski

doi:10.1371/journal.pone.0316331

. 2024 Dec 30;19(12):e0316331. doi: 10.1371/journal.pone.0316331

Evidence for gene essentiality in Leishmania using CRISPR

Wen-Wei Zhang ¹, Greg Matlashewski ^1,^*

Editor: Ben L Kelly²

PMCID: PMC11684651 PMID: 39775585

Abstract

The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.

Introduction

Leishmania protozoa are transmitted by infected sand flies and cause human leishmaniasis resulting in pathologies ranging from self-healing cutaneous lesions to lethal visceral infections [1]. There is no vaccine for human leishmaniasis, and existing treatments rely on drugs with toxic side effects and varying efficacy depending on the type of leishmaniasis. The development of an effective vaccine and new treatments remain the research priorities for control and eventual elimination of leishmaniasis [1]. To identify potential drug targets, develop attenuated vaccine strains and understand the biology of this parasite, it is necessary to determine the essentiality and function of Leishmania genes [2].

CRISPR (clustered regularly interspaced short palindrome repeats) is a gene-editing technology developed from a bacterial antiviral defense system that has been effectively applied to study genes in eukaryotic cells [3–7]. Cas9, an RNA guided endonuclease and a guide RNA (gRNA) are the two main components of CRISPR gene editing system. The gRNA uses its guide sequence to scan the genome and directs Cas9 nuclease to the specific complimentary DNA target site 3 base pairs upstream from a PAM site such as NGG to generate a double-stranded break (DSB). Repair of the DSB through homology directed repair (HDR) can be used to introduce DNA sequences with desired editing, mutations or selectable markers into the DSB site. Because of its relative simplicity, CRISPR gene editing has been widely used in a variety of organisms including Leishmania.

CRISPR gene editing has been adapted for use in Leishmania since 2015 and has greatly improved gene targeting efficiency in Leishmania [8–11]. However, since Leishmania does not have the functional nonhomologous end-joining (NHEJ) pathway due to lack of DNA ligase IV, in the absence of repair templates, Leishmania mainly uses Single Strand Annealing (SSA) or Microhomology Meditated End Joining (MMEJ) pathways to repair double strand breaks (DSB), which are not efficient and can lead to co-deletion of the target gene and its adjacent genes [9, 10, 12, 13]. Thus, repair template containing an antibiotic selection marker is often used to improve gene targeting specificity and facilitate isolating CRISPR targeted gene mutant [8, 9, 11].

Two CRISPR based protocols have been commonly used for gene editing in Leishmania including the plasmid based stable gRNA and Cas9 expression vector using rRNA gene promoter (rRNA-P stable protocol) [10] and the transient gRNA expression protocol using a T7 promoter (T7 transient protocol) [11]. In the rRNA-P stable protocol, the gRNA coding sequence is inserted into a plasmid vector expressing the Cas9 gene under the control of the Leishmania ribosomal RNA promoter and stable transfectants are generated [10, 12]. This stable gRNA and Cas 9 expression protocol has been used to generate gene disruption and deletion mutants, single point mutations, and to tag endogenous genes with or without using a selection marker. In particular, the rRNA-P stable protocol has been used to delete multicopy family genes, to reveal essentiality of various Leishmania genes by direct observation of dying null mutant cell clones [9, 10, 12–15], and to generate selection marker free attenuated Leishmania mutants as vaccine candidates [16–18]. In addition, the gRNA and Cas9 are expressed in a single CRISPR plasmid in rRNA-P stable protocol, making it convenient to carry out gene targeting in new Leishmania strains and clinical isolates [10, 19].

In the T7 transient protocol, a Leishmania strain is first engineered to constitutively express the Cas9 endonuclease and T7 polymerase, gene editing is achieved by transfecting this strain with linear DNA encoding the gRNA downstream from the T7 promoter sequence and donor DNA encoding antibiotic selection markers or fluorescent protein tags [11]. The gRNA template and donor DNA can be conveniently prepared by PCR [11]. The T7 transient protocol is more convenient and faster than the stable rRNA-P protocol once the Leishmania strain constitutively expressing Cas9 and T7 polymerase has been established, as it bypasses the requirement of construction, transfection and selection for the gRNA/Cas9 expression plasmid. The simultaneous transfection of two antibiotic selection marker donors makes it possible to isolate many of the gene null mutants with no need for cell cloning [11]. Thus far, hundreds of Leishmania genes have been successfully targeted with this T7 transient protocol and this has greatly expanded our understanding of Leishmania genes and biological systems [20–22].

Given that the gRNAs are transiently expressed in the T7 transient protocol [11], it is possible that Cas9 nuclease may require more time to target multiple copy genes or genes in multiploidy chromosomes even though the gene may be dispensable for Leishmania viability. Alternatively, gene null mutants with reduced proliferation may be more difficult to isolate using the T7 transient protocol due to omission of cell cloning [11, 20–22]. We therefore were interested to investigate whether the stable expression of gRNA may be more efficient at establishing null mutants for multicopy genes and genes which are required for optimal cell proliferation. The rRNA-P stable protocol was used to target 22 Leishmania genes that, in previous studies, the T7 transient protocol did not generate the live null mutants [20–22]. Notably, the rRNA-P stable protocol was able to generate live null mutants for 8 of these genes and provide evidence that the remaining 14 genes were essential by observing dying and death of the null mutant promastigote clones in culture. These results suggest that the rRNA-P stable protocol is a useful complement to the T7 transient system for investigating gene essentiality and may be more suitable for targeting multicopy genes.

Results

Non-essential genes

Initially, it was necessary to consider which Leishmania genes to include in this study. A list of L. mexicana and L. donovani genes in which null mutants were unable to be generated with the T7 transient protocol were selected as shown in Table 1 [20–22]. Using the rRNA-P stable protocol, we determined that it was likewise not possible to generate alive null mutants for 14 of these genes consistent with these genes being essential for promastigote survival (see S1 Fig and below). It was however possible to generate live null mutants for 8 of these genes with the rRNA-P stable protocol including for example the L. donovani LdBPK_100590 and LdBPK_230540 genes that encode hypothetical proteins of unknown function (Fig 1). As the general approach used in this study, Leishmania promastigotes were transfected with the pLdsaCN (or pLdCN) plasmid expressing Cas9 nuclease and a Ld100590 gene-specific gRNA followed by selection with G418 as outlined in Fig 1A and detailed in Methods. Resistant promastigotes were subsequently transfected with a linear donor DNA consisting of a PCR product containing the Bleomycin resistance gene with 25 bp homology arms to the Cas9 cleavage site followed by selection in culture with Bleomycin and G418. To prevent overgrowth of partially targeted cells and reduce the time to isolate the gene disruption mutants, the double resistance parasites were cloned in a 96 well plate and the cell proliferation status in each well was monitored every 2–3 days. If the gene to be targeted is non-essential, the cloned promastigotes would continue to proliferate. PCR analysis of 3 null clones shows a complete absence of smaller wildtype Ld100590 gene band with the larger band representing the targeted gene containing the Bleomycin resistance gene (Fig 1B). Likewise, 4 null mutants were generated with the rRNA-P stable protocol for the single copy L. donovani gene Ld230540 (Fig 1C).

Table 1. Comparison of two CRISPR methods in targeting Leishmania genes.

Gene ID¹	Gene Function	Chromosome	Gene copies²	T7 transient protocol		rRNA-P stable protocol		Required for viability
				Ref	WT gene	WT gene	Observation of dying cells⁵
				Ref	Retention³	Retention⁴	Observation of dying cells⁵
LmxM.02.0290	Mitogen-activated kinase kinase kinase	2	2	21	Yes	Yes	Yes	Yes
LmxM.03.0780	Serine/threonine-protein kinase	3	2	21	Yes	Yes	Yes	Yes
LmxM.08.0530	Protein kinase	8	2	21	Yes	Yes	Yes	Yes
LmxM.08_29.1330	Serine/threonine-protein kinase Aurora kinase 2, AUK2	8	2	21	Yes	Yes	Yes	Yes
LmxM.09.0910	Calmodulin	9	6	20	Yes	Yes	Yes	Yes
LmxM.16.1550	Component of motile flagella 6 (CMF6)	16	3	20	Yes	No	No	No
LmxM.17.0790	Polo-like protein kinase, PLK	17	2	21	Yes	Yes	Yes	Yes
LmxM.20.0960	Protein kinase	20	2	21	Yes	Yes	Yes	Yes
LmxM.20.1180	Calpain-like cysteine peptidase	20	2	20	Yes⁶	No	No	No
LmxM.24.2010	Phosphatidylinositol 3-kinase, PI3K	24	2	21	Yes	Yes	Yes	Yes
LmxM.25.2340	AGC essential kinase 1, AEK1	25	2	21	Yes	Yes	Yes	Yes
LmxM.30.2860	Tousled-like kinase, TLK	30	4	21	Yes	Yes	Yes	Yes
LmxM.30.2960	Repressor of differentiation kinase 2, RDK2	30	4	21	Yes	Yes	Yes	Yes
LmxM.34.3960	Protein kinase A catalytic subunit isoform 2, PKAC2	34	2	21	Yes⁶	No	No	No
LmxM.34.4010	Protein kinase A catalytic subunit isoform 1, PKAC1	34	2	20,21	Yes⁶	No	No	No
LdBPK_100590	Hypothetical protein	10	2	22	Yes	No	Yes⁷	No
LdBPK_111030	Hypothetical protein	11	2	22	Yes	Yes	Yes	Yes
LdBPK_230540	Hypothetical protein	23	2	22	Yes	No	Yes⁷	No
LdBPK_260650	Protein of unknown function, DUF2012	26	2	22	Yes	Yes	Yes	Yes
LdBPK_310120	FG-GAP repeat protein	31	4	22	Yes	No	Yes⁷	No
LdBPK_312380	3’-nucleotidase/ nuclease	31	4	22	Yes	No	No	No
LdBPK_354780	Hsp70 protein	35	2	22	Yes	Yes	Yes	Yes

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1. LmxM stands for L. mexicama; LdBPK stands for L. donovani.

2. These numbers do not include the additional gene copies present in the extra chromosome circles if exist.

3. Whether a wild type (WT) gene band was detected in the surviving transfectant population.

4. Whether a wild type (WT) gene band was detected in all surviving transfectants after cloning in a 96 well plate.

5. The dying and dead cells were caused by the disruption of all wild type gene allele present in these clones.

6. Likely because the primers used to verify the CRISPR deletion mutants were non-specific to the target gene, the WT size PCR band detected could as well be derived from the conserved sequence which are also present in the nearby gene (See S2 and S3 Figs). Indeed, Fochler et al have recently shown that LmxM.34.3960 and LmxM.34.4010 alive null mutants could also be generated with T7 transient protocol when the gene-specific primers were used to confirm the CRISPR deletion mutants [26].

7. Likely due to failure of quick adaptation, some dead cells could be observed from the transfectants when these important but non-essential genes were targeted by CRISPR.

Fig 1 — (A) Schematics of *Leishmania* CRISPR plasmid pLdSaCN and strategy used for gene disruption. In pLdSaCN CRISPR plasmid, L. *donovani* rRNA promoter (rRNAP) controls the stable transcription of the gRNA and *Staphylococcus aureus* Cas9 (SaCas9). The target gene specific gRNA leads Cas9 to generate a double strand break which is then repaired following the introduction of the donor DNA (black bars) containing an antibiotic resistance gene, resulting in disruption of the target gene. A2-IGS, A2 intergenic sequence; NEO, Neomycin resistance gene. Note, the pLdSaCN plasmid is structurally like the other stable expression *Leishmania* CRISPR plasmid pLdCN also used in this study. Instead, pLdCN expresses the commonly used *Streptococcus pyogenes* Cas9 (SpCas9). (B) PCR analysis with primers L and R showing the disruption of a non-essential L. *donovani* gene (LdBPK_100590). The higher disrupted gene band with the inserted donor DNA but not the lower WT gene band was detected in three Ld100590(-/-) clones. (C) Complete disruption of a non-essential L. *donovani* gene (LdBPK_230540).

Essential genes

To understand Leishmania biology and identify potential new drug targets, it is necessary to determine the essentiality of Leishmania genes [2]. To illustrate how gene essentiality is normally determined with the rRNA-P stable protocol, we use targeting AGC essential kinase 1 gene (LmxM.25.2340, AEK1) as an example (Fig 2). As shown in Table 1, null mutants were previously not generated for the AGC essential kinase 1 gene (LmxM.25.2340, AEK1) with the T7 transient protocol and this was the same outcome using the rRNA-P stable protocol consistent with this being an essential gene. As described above, promastigotes were transfected with pLdsaCN (or pLdCN) plasmid and selected with G418 followed by transfection of the Puromycin resistance gene donor. Ten to fourteen days later, the G418 and Puromycin double resistance parasites were cloned into a 96 well plate. Different from the T7 transient protocol, the constantly expressed Cas9/gRNA complex would continue to scan the genome and target the remaining copy or copies of the gene if still available after cloning. If the gene is required for viability following single cell cloning, once the remaining copy of the gene has been disrupted by CRISPR, promastigotes would stop growing or multiply slowly to form clumps until the gene products (mRNA and protein) are diluted and degraded to the minimum level required for survival, as shown for the LmxM.25.2340 null mutants in Fig 2A. Depending on the relative importance, initial abundance, stability of the gene product in the cell and the cloning time (stage) after the complete gene disruption for the individual clone, the dying (dead) cell clumps could contain only a few promastigotes to more than hundreds of promastigotes (Fig 2A, S1 Fig and below). PCR analysis of all the surviving clones in the 96 well plate will show the WT gene band persists and at least one allele of the essential gene was successfully targeted and disrupted by CRISPR (Fig 2B, S1 Fig and below) [9, 10, 12, 14]. In this manner, by combining observation of the death of gene null mutant clones and detection of the WT gene band and the gene targeting band in all surviving clones, using the rRNA-P stable CRISPR protocol, we were able to confirm that many of those genes for which the T7 transient protocol was not able to generate alive null mutants (14 out of 22; see Table 1, Figs 2, 3, and S1 Fig) are truly essential for Leishmania viability.

Fig 2 — (A) The dying promastigotes clumps observed in some of the 96 well plate wells after the G418 and Puromycin double resistance transfectants were cloned into a 96 well plate. Scale bar, 15 μm. (B) PCR analysis showing the wild type (WT) AEK1 gene band persists in all surviving clones in the 96 well plate and at least one of the gene alleles was correctly disrupted by CRISPR.

Fig 3 — (A) *Calmodulin* gene locus in chromosome 9 contains three *Calmodulin* genes (LmxM.09.0910; 0920 and 0930) in tandem array. (B) Strategy used by rRNA-P stable protocol to delete and disrupt L. *mexicana Calmodulin* gene clusters, and the outcome of one of the *Calmodulin* gene targeting clones. The PCR primers used to verify gene deletion and disruption are indicated. (C) PCR analysis of the *Calmodulin* gene targeting clone shown in B. After CRISPR gene targeting, the PCR product size with primers 10R1+30L1 reduced from 4.2 Kb of the WT band size to 2.8 Kb and no PCR product could be detected with primers 30R1+L, indicating LmxM.09.0930 gene had been deleted. The PCR product sizes of primers R+10 L1 increased from 700 bp to 900 bp and 1100 bp respectively, indicating both LmxM.09.0910 alleles had been disrupted. Both the WT band and size increased disruption band of primers R+30L1 were detected, indicating one LmxM.09.0920 allele had been successfully disrupted and one WT LmxM.09.0920 allele (the only WT *Calmodulin* gene allele) remained in this clone. Note: a smaller than the expected size (R+10R1) band (900 bp) was detected in this *Cal* (+—/—) clone, indicating a recombination deletion had occurred in one of the two disrupted LmxM 09.0910 alleles, which explains the additional fainter band running at 700 bp detected in the PCR with (R+L) primer pair. Likewise, the fainter band (the middle band) detected in the PCR with (R+30L1) primer pair could be derived from some of the cells in this *Cal* (+—/—) clone cell population where recombination deletion took place in the disrupted LmxM 09.0920 allele. (D) The dead cell clumps observed after the CRISPR gene targeting transfectants were cloned into a 96 well plate, suggesting all the *Calmodulin* genes in the dead clones had been deleted or disrupted. (E) Compared with the WT promastigotes, one of the surviving *Calmodulin* CRISPR gene targeting clones (+—/—) in the 96 well plate is shown with a smaller and round morphology. Scale bar, 15 μm.

As shown in Table 1, the rRNA-P stable protocol and the T7 transient protocol provide evidence that the LmxM.09.0910 encoding a calmodulin analog is an essential gene. Calmodulin is a highly conserved Ca²⁺ binding protein present in all eukaryotic cells that relays signals to various calcium-sensitive enzymes, ion channels and other proteins. Leishmania contains 3 copies of calmodulin gene (LmxM.09.0910, LmxM.09.0920 and LmxM.09.0930) in tandem array in chromosome 9 (Fig 3A). The strategy used by the rRNA-P stable protocol to target the L. mexicana calmodulin gene family and one of its outcomes are illustrated in Fig 3B. The pLdsaCN plasmid expressing a single gRNA targeting all 3 calmodulin genes was transfected followed by the Bleomycin resistance gene donor. As expected, dying (dead) clones were observed in some wells after the transfectants were cloned (Fig 3D and S1 Fig). PCR analysis showed that at least one copy of the wild type of Calmodulin gene was retained in all surviving clones (S1 Fig). Further analysis of the slowest growing clone revealed (see detailed explanation in Fig 3 legend) CRISPR gene targeting had deleted both alleles of LmxM.09.0930, disrupted both alleles of LmxM.09.0910 and one allele of LmxM.09.0920, and only one wild type LmxM.09.0920 allele remained (Fig 3B and 3C). The promastigotes of the slowest growing clones were less motile and more rounded than wild-type promastigotes (Fig 3E and below). These data are consistent with the previous report [20] providing evidence that like in other organisms, Calmodulin is essential for Leishmania. It is also interesting to point out that Calmodulin is the only one of the 98 Leishmania flagellar protein genes which is required for Leishmania viability [20].

Genes on polyploid chromosomes

Genes present on Chromosome 31 have at least 4 copies since this chromosome is tetraploid. For example, the LdBPK_310120 (FG-GAP repeat protein) and the LdBPK_312380 (3’NT/NU) genes are located in chromosome 31 [22]. As indicated in Table 1, it was not possible to delete all the copies of these genes using the T7 transient protocol. We attempted to generate null mutants in these genes using the rRNA-P stable protocol as described above. As shown in Fig 4A, expression of a single gRNA followed by introducing a donor DNA encoding the Bleomycin resistance gene generated clones with the wildtype gene replaced with the larger migrating genes containing the donor DNA and no remaining wildtype gene. This demonstrates that the LdBPK_310120 and LdBPK_312380 genes could be disrupted with constant expression of gRNA and Cas9 to generate null mutants.

Fig 4 — (A) CRISPR disruption of L. *donovani* genes LdBPK_310120 and LdBPK_312380 in the tetraploid chromosome 31. A total of four independent Cas9 cleavage and insertion events were required to disrupt all four copies of the genes (see left panel). (B) Disruption of the L. *mexicana* LmxM.16.1550 (CMF6) gene family in the trisomic chromosome 16. Three Cas9 cleavage and insertion events were required to disrupt all three copies of the genes (see left panel). Note, the LmxM.16.1550 gene was completely disrupted in only two of the 13 CRISPR gene targeting clones examined, highlighting the importance of cloning to isolate gene disruption mutants.

In another example of a non-essential gene, the L. mexicana LmxM.16.1550 gene (Component of motile flagella 6) (Table 1) is in chromosome 16 which is trisomic in L. mexicana. As shown in Fig 4B, it was possible to generate 2 null clones (-/-) with all three copies of the wildtype LmxM.16.1550 gene disrupted with the integrated donor DNA. There were however more clones displaying partial gene targeting (+/-) suggesting there is pressure to retain this gene. This demonstrates that it is advantageous to perform cell cloning following gene targeting to isolate some homozygous gene mutants with multiple copies or clones that are slower growing.

Genes required for optimum proliferation

If a gene product is required for the optimal proliferation of promastigotes, the homozygous gene null mutants could be outcompeted in culture by the heterozygous mutants in the population of CRISPR edited transfectants. This would result in loss of the null mutant unless the mutant was cloned. To investigate the possibility that some null mutants may be slow growing, we compared the proliferation of the null mutants generated in this study. As shown in Fig 5, the proliferation rates of the null mutants LdBPK_100590 and LmxM.16.1550 were much slower than the wildtype promastigotes. Thus, cell cloning would be necessary for isolation of mutants with reduced proliferation such as for example the LmxM.16.1550 mutant clones where a relatively low percentage of null mutants was detected in the above PCR analysis (Fig 4B).

Fig 5 — Equal numbers of *Leishmania* promastigotes (1 million promastigotes per ml) were inoculated in flasks each containing 4 ml culture medium. The parasite growth was monitored by microscope counting once a day for 8 days. The data shown are the mean plus Standard Error of the Mean (SEM). Note, as examples, the cell density differences between LdWT and Ld100590(-), Ld230540(-), Ld310120(-) and Ld312380(-) cells, and between LmxWT and Lmx161550(-) cells at day 3 post inoculation are statistically significant (Student’s t-test P<0.001). This is the representative data of three independent experiments.

Phenotypic changes in gene disrupted mutants

Mutation of flagellar protein encoding genes can affect the motility of L. mexicana promastigotes in culture [20] and 3 of the L. mexicana flagellar genes listed in Table 1 (16.1550, 20.1180, 34.4010) were identified as non-essential using the rRNA-P stable protocol in this study. We therefore examined whether the mobility of these mutants was affected using a mobility assay described in Methods. As shown in Fig 6, the LmxM.16.1550 (Component of motile flagella 6) and Lmxm.34.4010 (PKAC1) mutants were largely defective in swimming forward compared to wildtype or the LmxM.20.1180 mutant (S1–S3 Movies). The forward swimming assay showed the number of promastigotes reaching the opposite end of the tube at different time points was significantly reduced for the LmxM.16.1550 and LmxM34.4010 null mutants. Notably, near 50% of LmxM.16.1550 null mutants kept turning around in circles (S2 Movie) despite the flagellar length appeared to be normal (Fig 6B). The LmxM.34.4010 deficient promastigotes in contrast were largely motionless or wiggled in a slow speed despite the flagellar length and promastigote size appearing normal (S3 Movie and Fig 6B). The Calmodulin gene targeting mutant [LmxM.09.0910 (+—/—)] retaining at least one wildtype gene was smaller in size and swam more slowly in culture medium (S4 Movie and Fig 6). In comparison, the LmxM.20.1180 (CALP1.1) null mutants are normal in size but appeared to be able to proliferate and swim slightly faster than the wild type cells (S5 Movie and Figs 5 and 6).

Fig 6 — (A) A swimming assay was performed on a 7 cm VINYL Tubing with both ends in an up position as described in methods. The tube was first filled with 900 μl PBS, then 2 million L. *mexicana* promastigotes in 50 μL culture medium were gently loaded at the left end of the tube. The data shown in the table are the mean number (plus Standard Deviation, SD) of promastigotes detected in 3 μL solution taken from the right end of the tube after incubation for 1, 1.5, 2 and 3 hours. The formalin fixed L. *mexicana* promastigotes (Dead LmxM WT) was used as the negative control. The differences of promastigote number detected in the 3 μL solution between L. *mexicana* WT and the various mutant cell lines are statistically significant (Student’s t-test, * P<0.05; ** P< 0.005; *** P< 0.0005; ******** P< 0.0001). (B) The Giemsa-stained L. *mexicana* promastigotes. The promastigote size and its flagellar length appear normal for the null mutants of LmxM.16.1550, LmxM.34.4010 and LmxM.20.1180. However, the promastigotes of *Calmodulin* CRISPR gene targeting clone (+—/—) are smaller than WT L. *mexicana* promastigotes though the normal flagellar lengths are still retained. Scale bar, 5 μm.

Discussion

It is important to identify essential genes to define biochemical pathways that represent potential intervention targets [2], and this can be performed in Leishmania using CRISPR based methodologies. This study demonstrates the ability of the rRNA stable protocol to provide evidence for gene essentiality. The advantage with the T7 transient protocol is that it omits the requirement to clone a gRNA encoding sequence in the pLdCN plasmid and transfection making it more practical for high throughput gene targeting. The importance of performing high throughput gene deletion cannot be overstated since the mutants provide a wealth of information on the biology of Leishmania.

More than 5% of Leishmania genes have multiple copies present either in aneuploid chromosomes or in tandem array [23, 24], and this study contends that the rRNA stable protocol is more suitable for generating multi-copy gene null mutants. The Trypanosoma brucei RNAi library screen study revealed that at least 10% T. brucei genes are essential for viability and 25–37% of genes are required for optimal growth under various culture conditions [25]. An advantage of cloning null mutants is the potential to directly visualize dying promastigotes by microscopy such as for example with the AEK1 null gene mutant (Fig 2A) and the ability to isolate slower growing clones. At least one copy of the wildtype essential AEK1 gene allele can be detected by PCR in the remaining surviving clones as seen in Fig 2B.

A five-star methodology approach to establish gene essentiality in Leishmania has been proposed which we are in general agreement with [2]. In practice however, it is difficult to meet the most stringent criteria using forced plasmid shuffling and DiCre gene deletion that have so far only been applied to a few Leishmania genes [2]. Regardless of how essential genes are classified, this study highlights that the rRNA stable protocol should be considered when it is not possible to generate a null mutant with the T7 transient protocol. In addition to providing evidence for gene essentiality, the ability to generate null mutants is necessary for studying differentiation, virulence, pathogenesis and basic biology of the parasite.

During our study, we have also noted that when targeting a single member of a multicopy gene family, it is important to select PCR primers specific to the targeted gene that do not also amplify other members of the gene family. This is important for example with the PKAC1 (LmxM.34.4010) and PKAC2 (LmxM.34.3960) gene family members that have similar but distinct sequences (see S2 and S3 Figs). Otherwise, even though the gene has been successfully targeted, the PCR analysis of that gene will show that the wild type gene has been retained. Consistent with the above observations, the generation of L. mexicana PKAC1 and PKAC2 null mutants with the T7 transient protocol has recently been reported using gene-specific PCR primers to verify the deletion mutants, and a similar phenotype as this study for the PKAC null mutants was observed [26].

The LeishGEM (Leishmania genetic modification project) is in progress (http://www.leishgem.org/) and represents an important resource to the Leishmania field. As this is a high throughput project to develop null mutants for all non-essential genes, it is necessary to use the high throughput T7 transient protocol [11]. The rRNA-P stable protocol may however be considered as a secondary approach when null gene mutants cannot be generated with the T7 transient protocol such as for example multi-copy genes or slow growing null mutants. Although it may be possible to generate slow growing null mutants using the rRNA-P stable protocol, it is nevertheless possible that the targeted gene may still be considered essential if compensating genome alterations take place to allow the null mutant to survive. Compensating genetic changes such as for example amplification of a different gene may be identified by whole genome sequencing and analysis of these genes can provide important insight into the function or biological pathways of the targeted genes.

CRISPR base editing has been recently adapted for use in Leishmania to introduce STOP codons in targeted genes [27]. Base editing differs from gene editing as it bypasses the DNA double strand break repair pathways, thus it requires no repair DNA donors and has significantly increased the gene inactivation efficiency through introduction of a premature STOP codon within coding sequence [27]. This advancement makes it feasible to construct the Leishmania loss of function CRISPR base editing libraries, though small sub-libraries may be required to overcome low transfection efficiency, and the extended culture time needed to generate the edited mutants. It is noteworthy that the plasmid vector used to perform base editing in Leishmania (pLdCH-hyBE4max) uses the pLdCH plasmid used in the rRNA-P stable protocol with the Cas9 gene replaced by the base editing Cas9 fusion gene (hyBE4max) [27]. Similar to the rRNA-P stable protocol described within, the base editing study reported that stable expression of gRNA and hyBE4max and prolonged culture were required for more efficient base editing [27].

In summary, for the large majority of Leishmania genes, the T7 transient CRISPR protocol is highly effective to generate null mutants, and this is particularly advantageous for high throughput gene targeting. The rRNA-P stable CRISPR protocol is highly effective to generate null mutants of genes with multiple copies and slow growing mutants. The more recent development of loss of function base editing will further expand the CRISPR technologies available for studying the Leishmania genome. Collectively, these complementary approaches have the potential to generate a wealth of knowledge about the function of the over 8000 genes in Leishmania genome for the development of novel treatments, vaccines and diagnostic tests.

Materials and methods

Leishmania strains and culture medium

Leishmania mexicana (MNYC/BZ/62/M379) and L. donovani 1S/Cl2D promastigotes were cultured at 27°C in M199 medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum, 40 mM HEPES (pH 7.4), 0.1 mM adenine, 5 mg l⁻¹hemin, 1 mg l⁻¹ biotin, 1 mg l⁻¹ biopterine, 50 U mL⁻¹ penicillin, and 50 μg mL⁻¹ streptomycin. Leishmania promastigotes were passaged to fresh medium at a 40-fold dilution once a week.

gRNA design and cloning

The gRNAs were designed with the aid of Eukaryotic Pathogen CRISPR guide RNA Design Tool (EuPaGDT) (http://grna.ctegd.uga.edu/) to avoid off target sites in the genome. A single gRNA guide coding sequence was ordered as standard oligos with 5′-TTGT and 5′-AAAC overhangs. All oligos and primers used in this study were ordered from Alphaadn (http://alphaadn.com/) or IDT (https://www.idtdna.com). The optimal guide length is 19 or 20 nt for SpCas9 gRNA and 21 nt for SaCas9 gRNA. After phosphorylation and annealing, the gRNA guide coding sequences were ligated into the Bbs I site of pLdCN or pLdSaCN CRISPR vector as described [19]. All oligos and primers used in this study are listed in S1 Table.

Parasite transfection

4 × 10⁷ Leishmania promastigotes (middle log phase to early stationary phase) were harvested and washed once in 200 μL Tb-BSF buffer (90 mM Na₂HPO₄, 5 mM KCl, 0.15 mM CaCl₂, 50 mM HEPES, pH 7.3), and resuspended in 100 μL Tb-BSF buffer. Then, 2 to 5 μg CRISPR plasmid vectors in a volume < 20 μL were added and mixed. The transfection was performed in a 2-mm gap electroporation cuvette with the LONZA Nucleofector 2b Device (program U33). The transfected Leishmania cells were selected with 100 μg/mL G418 in the following day and selection takes 10 to 14 days to establish a resistant culture. Once the CRISPR plasmid vector transfected cell culture was established, those cells were then transfected with the antibiotic selection marker donor which was prepared by PCR and contains 25 bp homology arms to the Cas9 cleavage site. In the following day, 100 μg/mL Zeocin, or 30 μg/mL puromycin was added into the donor transfected cell culture. After 5 to 6 days incubation, the medium was replaced once with fresh medium before cloning into a 96 well plate. Note: The bleomycin resistance gene cassette conveys resistance to zeocin; the Puromycin resistance gene cassette conveys resistance to Puromycin.

Cloning into 96-well plates

To prevent overgrowth of partially targeted Leishmania promastigotes in the culture flask, the double antibiotics (G418/ Zeocin or Puromycin) resistant promastigotes were cloned into a 96 well plate once enough cells were available for cloning, usually 10 to 21 days post donor transfection. Since Zeocin takes at least one to two weeks to completely kill the wildtype parasites, we waited about two weeks before cloning the bleomycin resistance gene donor transfected cells. After cloning into a 96 well plate, cell proliferation in each well of the plate would be monitored every two to three days. More attention was paid to the slow growing clones, which were then marked to facilitate following up. Since disruption of all the essential gene alleles would eventually result in cell death, cells in those wells could replicate slowly initially then stop growing once the gene product was diluted and degraded to the minimum level required for cell survival. If the gene product is required for optimal growth but non-essential, cells in those slow growing wells were most likely to be the gene null mutant clones. Fewer surviving clones will be observed after cloning into a 96 well plate when targeting essential genes. Depending on the gRNA activity, the relative importance and abundancy of the essential gene product, 3–15 dying clones can usually be observed after the double antibiotic resistance transfectants are cloned into a 96 well plate. The dying clump size could vary depending on the relative importance and abundance of the essential gene product. More dying clones may be observed when large size dying clumps exist in a 96 well plate. It is also possible that more dying clones will be observed when an essential gene is targeted with a gRNA with higher activity.

If no dying clones was observed in the 96 well plate, 6–10 clones (especially the slow growing clones) were initially selected for PCR analysis. If necessary, more clones could be analyzed until a null mutant clone was detected. If many dying clones and empty wells were already observed in the 96 well plate, ideally all the remaining surviving clones or at least the 20 slower growing clones in the plate should be PCR analyzed to determine the gene essentiality.

Genomic DNA preparation and PCR analysis

Parasite genomic DNA was extracted from Leishmania promastigotes with the minipreparation method as described [28]. The purity and quantity of those genomic DNA were assessed by Nanodrop spectrophotometer. Parasite genomic DNA could also be prepared with M-Fast PCR Genotyping kit (ZmTech Scientifique, Montreal). Briefly, 100 μL stationary phase Leishmania culture was harvested by centrifugation at 1,500 g for 5 min, and the cell pellet was resuspended in 15 μL reagent-A and incubated at 95°C for 30 min in a PCR apparatus. Once the tube cooled to room temperature, 3 μL reagent-B was added into the parasite tube and mixed well. Then, 0.5 μL lysate supernatant was added into 11.5 μL PCR mastermix for a total 12 μL reaction.

Primers were designed manually or using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/). Optimal primer length was 20 nucleotides with 60°C Tm. The Taq DNA polymerases used in this study include 2X DreamTaq Green PCR Master MIX, and 2X Platinum SuperFi PCR Master Mix (Thermo Fisher Scientific). The PCR program was set up according to manufacturer’s instruction with variation in annealing temperature, extension time, and PCR cycles. The PCR products were separated in 1% to 1.5% agarose gel. If required, the specific PCR bands were extracted from the gel and sent to Genome Quebec Sequencing Center for sequencing confirmation.

Growth curves and cell imaging

Leishmania promastigotes were seeded at the density of 1 × 10⁶ promastigotes/mL in 4 mL M199 media in 25 cm² flasks and counted by a microscope daily for 8 days. A minimum of three biological replicates were evaluated. Movies and dead cell clumps were recorded with Nikon ECLIPSE TE200 Inverted Microscope and Nikon superhigh-performance 3 x zoom digital camera COOLPIX990. The Giemsa-stained L. mexicana promastigotes were examined with an OLYMPUS BH-2 light microscope and photographed with OMAX A35140U 14MP USB2.0 C-Mount Microscope Camera.

Promastigote motility assay

A promastigote swimming assay was performed on a 7 cm vinyl Tubing (3/16 ID x 1/16 wall) with both ends in up position after turning the tube near 90 degrees at the ends in an Eppendorf tube rack (see Fig 5A). The tube was first filled with 900 μl PBS, then 2 million L. mexicana promastigotes in 50 μL culture medium were gently loaded at the left end of the tube. After incubation at room temperature for 1, 1.5, 2 and 3 hours, 3 μL solution was taken from the right end of the tube for detecting with a microscope and quantitation of promastigotes reached the right end of the tube.

Supporting information

S1 Fig. Evidence for Leishmania essential genes included in Table 1 using the rRNA-P stable CRISPR protocol.

PCR shows the retention of the wild type gene for all surviving clones tested and at least one allele of the essential gene in the surviving clones (+/-) was successfully targeted and disrupted by CRISPR. Observation of the dying and dead promastigotes clumps of the essential gene null mutants’ clones (-/-) in 96 well plates. In the rRNA-P stable CRISPR protocol, the gRNA and Cas9 are constantly expressed, the gRNA/Cas9 complex will continue to scan the genome until the last copy of the target gene is deleted or disrupted. If the gene is required for viability, once the remaining copy of the essential gene has been disrupted by CRISPR after cloning into a 96 well plate, null mutants in those wells would stop growing or multiply slowly to form clumps until the gene products are diluted and degraded to the minimum level required for survival. Depending on the relative importance, and initial abundance of the essential gene product in the cell and how soon the individual promastigote was cloned into a 96 well plate after the complete gene disruption, the dying (dead) cell clump size could vary from only a few cells to more than hundreds of cells, the earlier the complete gene disruption took place before cloning, the smaller the size of the dying cell clumps was expected. In this approach, by combining observation of the dying gene null mutant clones and detection of the WT gene band in all surviving clones, using rRNA-P CRISPR protocol, we provide evidence that 14 out of 22 genes listed in Table 1 are essential for promastigote viability. Those essential genes include ten L. mexicana kinase genes: LmxM.02.0290 (Mitogen-activated kinase kinase); LmxM.03.0780 (serine/threonine-protein kinase); LmxM.08.0530; LmxM.08_29.1330 (serine/threonine-protein kinase; Aurora kinase 2, AUK2); LmxM.17.0790 (polo-like protein kinase, PLK); LmxM.20.0960; LmxM.24.2010 (phosphatidylinositol 3-kinase, PI3K); LmxM.25.2340 (AGC essential kinase 1, AEK1); LmxM.30.2860 (Tousled-like kinase, TLK); LmxM.30.2960 (Repressor of differentiation kinase 2, RDK2) and the Calmodulin gene LmxM.09.0910; and three L. donovani genes LdBPK_111030 (hypothetical protein); LdBPK_260650 (Protein of unknown function (DUF2012)) and LdBPK_354780 (Hsp70 protein) (also see Figs 2 and 3).

(PDF)

pone.0316331.s001.pdf^{(1.3MB, pdf)}

S2 Fig. Successful gene disruption of non-essential L. mexicana PKAC1 (LmxM.34.4010) and PKAC2 (LmxM.34.3960) with rRNA-P stable protocol.

(A) PKAC1 (LmxM.34.4010) and PKAC2 (LmxM.34.3960) genes are located in chromosome 34 and share large part of conserved sequences. (B) Strategy used by rRNA-P stable protocol to disrupt LmxM.34.3960 and LmxM.34.4010 genes. Use targeting PKAC1 as an example, a gRNA was designed to target the gene specific 5’ end coding sequence of PKAC1 gene, which was then disrupted with the bleomycin resistance gene donor. (C) PCR analysis showing both PKAC1 gene alleles were successfully disrupted. (D) PCR analysis showing both PKAC2 gene alleles were successfully disrupted.

(PDF)

pone.0316331.s002.pdf^{(68.6KB, pdf)}

S3 Fig. Successful gene disruption of L. mexicana CALP1 (LmxM.20.1180) with rRNA-P stable protocol.

(A) CALP1 (LmxM.20.1180) gene is located in chromosome 20 and shares the conserved sequence with the downstream CALP2 (LmxM.20.11185) gene. (B) Strategy used in this study to disrupt CALP1 gene. On the left panel, a gRNA was designed to target the specific 5’ end coding sequence of CALP1 gene, which was then disrupted with the bleomycin resistance gene donor. On the right panel, PCR analysis shows both CALP1 gene alleles were successfully disrupted.

(PDF)

pone.0316331.s003.pdf^{(51.6KB, pdf)}

S1 Table. Oligonucleotides and primers used in this study.

(DOCX)

pone.0316331.s004.docx^{(29.9KB, docx)}

S1 Movie. Leishmania mexicana wildtype promastigotes in culture.

(MOV)

pone.0316331.s005.MOV^{(10.8MB, MOV)}

S2 Movie. LmxM.16.1550 (CMF6) null mutant promastigotes in culture.

(MOV)

pone.0316331.s006.MOV^{(10.5MB, MOV)}

S3 Movie. LmxM.34.4010 (PKAC1) null mutant promastigotes in culture.

(MOV)

pone.0316331.s007.MOV^{(11MB, MOV)}

S4 Movie. LmxM.09.0910 (Calmodulin) partial deletion mutant (+—/—) promastigotes in culture.

(MOV)

pone.0316331.s008.MOV^{(11MB, MOV)}

S5 Movie. LmxM.20.1180 (CALP1.1) null mutant promastigotes in culture.

(MOV)

pone.0316331.s009.MOV^{(10.9MB, MOV)}

S1 Raw images

(PDF)

pone.0316331.s010.pdf^{(480.1KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0316331.r001

Decision Letter 0

Ben L Kelly

28 Aug 2024

PONE-D-24-31151Determination of gene essentiality in Leishmania using CRISPRPLOS ONE

Dear Dr. Zhnag,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

At the heart of this work is a comparison of null-mutant generation between two different CRISPR methodologies, the transient T7 approach and the stable rRNA promoter-driven gRNA approach. It is interesting and somewhat informative that in several instances, the stable approach has successfully yielded null mutants, where the transient approach targeting the same gene was unsuccessful. However, this work would have been stronger if the authors performed a direct, side-by-side comparison of the two techniques instead of comparing their stable approach data to the published outcomes of transient-based methodologies. This point is discussed in detail by Reviewer 2.

Additional points:

In line with concerns raised by Reviewer 2, the authors conclude that “dying and dead cells were caused by the disruption of all wild type gene allele present in these clones”, however presumably due to the obvious difficulties inherent with this, they do not provide direct supportive evidence.

The LmxM.20.1180 null mutant was described in the text as normal in mobility, yet the swimming assay data in Fig 5B indicate that it is more than twice as mobile as WT. This should be accounted for.

In Fig. 2D, please account for the additional fainter band running at 700 bp in the Cal (+--/---) lane.

Many minor issues with language throughout the manuscript that need to be edited.

In addition to these points, please also address all points raised by Reviewer 2.

==============================

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Reviewer #2: Partly

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Reviewer #1: N/A

Reviewer #2: No

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Reviewer #1: In this paper, Wen-Wei and Matlashewski investigate CRISPR as a tool for identifying essential genes in Leishmania. They compare the two commonly used CRISPR gene targeting methods in Leishmania: the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase-based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). The authors set to determine whether the plasmid-based rRNA-P stable protocol could generate viable gene null mutants that were previously considered essential using the T7 transient system. Here, the rRNA-P stable protocol was used to target 22 Leishmania genes previously considered essential using the T7 transient protocol. Notably, the rRNA-P stable protocol generated surviving null mutants for 8 of the 22 genes and confirmed essentiality for the remaining 14 genes. The authors indicate that this study demonstrates the advantage of performing the rRNA stable protocol to confirm gene essentiality that can be performed alone or following high throughput gene targeting with the T7 transient protocol to identify candidate essential genes. The results from this study suggest that the rRNA stable protocol is more suitable for generating multi-copy gene null mutants and null mutants with reduced proliferation because the gRNA and Cas9 are stably expressed from the CRISPR plasmid and the transfectants are cloned. The authors conclude that for the majority of Leishmania genes, the T7 transient CRISPR protocol is highly effective in generating null mutants, and this is particularly advantageous for high throughput gene targeting. The rRNA-P stable CRISPR protocol is highly effective in generating null mutants of genes with multiple copies and a slow-growing phenotype that is particularly advantageous to confirm gene essentiality. Furthermore, the recent development of loss of function base editing will further expand the CRISPR technologies available for studying the Leishmania genome. Collectively, these complementary approaches have the potential to generate a wealth of knowledge about the function of the over 8000 genes in the Leishmania genome for the development of novel treatments, vaccines, and diagnostic tests.

This is a timely and very important paper that provides important information on one of the most used techniques: gene deletion. Moreover, we, the scientists in the field of leishmaniasis, struggle to be sure about the essentiality of the gene we delete from the parasite genome. To date, CHRISPR is the most used technology for gene deletion. This study provides an important and useful guide on this topic. The paper is well written and describes a carefully designed study. Hence, this paper should be accepted for publication as is.

Reviewer #2: This paper reports the results of gene deletion attempts with a specific CRISPR protocol (named rRNA-P “stable” protocol). 22 genes were targeted, which in previous studies that used a different CRISPR protocol (“transient” protocol) did not yield confirmed null mutants. The authors show that they successfully knocked out some of these genes, and for some they also failed to obtain null mutants.

The stated aim, broadly, is to determine whether the “stable” protocol could be used to “define genes as essential in Leishmania”. The authors conclude that “This study demonstrates the advantage of performing the rRNA stable protocol to confirm gene essentiality […].” (line 128) This conclusion is flawed. Fundamentally, failure to generate a viable knock-out is never sufficient to prove that said gene is “essential”. Jones et al. 2018 (PMID: 29384366) have provided the most comprehensive analysis to date of reverse genetically engineered knockout mutants in Leishmania. They rehearsed in detail how a combination of different genetic approaches can increase the confidence with which a claim of “essentiality” can be made. That landmark study was not cited, but these considerations are highly relevant.

The attempts to compare the “stable” and the “transient” methods are flawed too. Using the two methods in parallel to target the same genes and compare results would allow for a comparison. Instead, results were taken from studies with different goals (generating large numbers of KOs, prioritising throughput) and compared here against the goal of isolating null mutants for a small handful of genes. This is a comparison of two very different experimental workflows from which few conclusions can be drawn about the relative power of these methods to achieve gene knockouts. The key difference is clonal vs. population analysis, rather than “stable” vs. “transient” CRISPR methods.

A clarification is required about the “stable strategy”: Cells transfected with a plasmid that contains both Cas9 and the gRNA were selected for a period of time. How much time – several days, a week? During this time, gRNA-complexed Cas9 will presumably cut the target locus. Some alleles will be mutated, many will be repaired though homologous recombination with the other allele, but can be cut again. What is the status of the target locus after stable expression of the Cas9-gRNA plasmid, but BEFORE introduction of the donor cassettes? The worry is that non-lethal mutations in the target locus, or reduced gene dosage, could promote adaptation to the loss of gene function. This would facilitate a subsequent full deletion. This is another marked difference in protocol, which makes comparisons between the methods, as presented, additionally challenging.

There is no doubt that different CRISPR protocols have different strengths and weaknesses and they should be used in complimentary ways. Particularly in situations of multi-copy gene families “stable” strategies, such as the one described here, will be invaluable. This is well elaborated in the discussion.

For the data itself, the conclusions form the successful gene deletions with the “stable” method are sound. The conclusions from the unsuccessful attempts are not all that well supported; important controls are missing.

Figure 1 shows that two genes that did not yield KOs in Ref 21, which used the “transient” method were successfully replaced by the “stable” method. The data showing loss of the WT band is sound.

Figure 2B. The death of clonal lines is taken as evidence of successful gene deletion. This is perhaps a reasonable assumption, but not proven. Death of the cells does not in itself “indicate all the calmodulin genes [..] had been deleted or disrupted” as stated in line 224. Observing that cells subjected to a CRISPR protocols ended up dead is neither proof of gene deletion (the DNA was not analyzed), nor of gene “essentiality” (ref. Jones et al. 2018). The level of evidence is the same whether this death occurs quickly (in the “transient” protocol) or slowly, as shown here with the “stable” protocol.

Figure 2D. The evidence that this clone has both WT and modified calmodulin loci is sound. The conclusion that the genotype is +-- / --- does not follow from these data. The PCR is not quantitative, the ratio of WT vs. modified copies cannot be estimated. Even the possibility that the locus has been amplified (perhaps with a point mutation in the sgRNA targeting site) cannot be excluded. Minimally, the statement “one of the calmodulin genes remains intact” (line 228) should be changed to “at least one…” (as done correctly in line 292). In short, whole genome sequencing of this clone should be done to clarify what the locus looks like in this clone.

It is also unclear how stable this genotype is, given the dynamic nature of the Leishmania genome. The quality of the microscopy images in Figs 2C-D is poor, but these round cells look markedly different from the flagellated ones shown in Figure 5B. Additionally, the growth curve in Fig 4 shows they grow at the same rate as WT. This would be surprising given the massive effect on morphology. This cell line could be valuable for studying the functions of calmodulin (outside of the scope of this paper). As it is, the presented data lacks sufficient information about the modified gene locus to conclude much, except that this clone is not a null mutant. This is largely the same conclusion that was drawn from the incomplete deletion of this gene by the “transient” method. Whether this failure to remove the gene completely is technical or biological (“essential” gene) cannot be proven by either method, without additional corroborating evidence.

Figure 3. Knockout of genes on supernumerary chromosomes.

Gene on chromosome 31 – the PCR result looks convincing.

CMF6. All alleles of the gene were lost in some clones following the “stable” protocol. The gene was removed in a minority of the tested clones. This conclusion is supported by the PCR result. The statement that presence of the gene in many clones indicates pressure to retain it is not strongly supported by the evidence. An alternative explanation is that the failure is technical: there could be sequence variation at this locus (perhaps more likely in a trisomic chromosome?) preventing recognition by the gRNA. Or the repair events that happened while cells had the Cas9 plasmid (before addition of the donor DNA) led to small sequence changes that made some alleles refractory to subsequent deletion. The sequence of the remaining allele should be determined to exclude these possibilities. The observation that the null mutants grew slower, is the most convincing evidence supporting the idea that the gene is required for normal growth.

Referring to Line 248 (also 265 ff): The idea that cloning the cells after transfection helps to isolate the null mutant from such mixed populations cases is correct. This is not a new idea and it is equally applicable to slow (“stable”) or fast acting (“transient”) gene deletion protocols. Beneke and Gluenz already published a detailed protocol for clone selection after transfection with the “transient” protocol in 2019 (PMID: 30980304).

Figure 5. The swimming assay in A lacks a negative control that cannot swim. An immobilized WT or a paralyzed or aflagellate mutant could be used to show how passive movement impacts on the recovery of cells from the other side of the tube. These data in themselves may indicate differences in motility, but are not compelling as presented. The results for PKAC1 are however consistent with the data by Fochler et al. Biorxiv 2023 that showed (i) that it was possible to isolate a PKAC1 null mutant also with the “transient” method, and (ii) this mutant was defective in flagellar beating.

Other comments:

The study design is based on the statement that the selected genes had been targeted unsuccessfully for knockout by CRISPR in one of three previous studies (refs 19-21 in the paper). The claim that these were “considered to be essential” is incorrect and should be modified, especially with regards to Ref 19, which did not comment on “essentiality” of any genes.

Consideration should be given to the biological functions of the targeted genes. This information should be added to Table 1.

line 229, “Additional PCR primers (not shown) had been used to verify the gene targeting outcome shown”. Please clarify what this means and include the relevant data or remove the statement.

Line 244, “triploid” means an extra set of chromosomes. Here, the correct term to describe the extra copy of chromosome 16 is “trisomic”.

Figure 4 please plot growth curves of exponentially growing cells on a semi-logarithmic graph (Y-axis on a log scale). Were doubling times calculated and which differences were significant compared to WT?

line 304 “its flagellum length appeared normal” – how was this quantified?

Line 439 – “dying crump” - do you mean clump?

Figure 5. The table in A lacks information. Do the numbers really represent numbers of Leishmania in 3µl (i.e. there were samples that contained 1 parasite)? How many times was each mutant measured? What statistical tests were done to support the statement that some mutants were slower?

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PLoS One. 2024 Dec 30;19(12):e0316331. doi: 10.1371/journal.pone.0316331.r002

Author response to Decision Letter 0

4 Oct 2024

Responses to Editor Dr. Kelly

Editor Comment. At the heart of this work is a comparison of null-mutant generation between two different CRISPR methodologies, the transient T7 approach and the stable rRNA promoter-driven gRNA approach. It is interesting and somewhat informative that in several instances, the stable approach has successfully yielded null mutants, where the transient approach targeting the same gene was unsuccessful. However, this work would have been stronger if the authors performed a direct, side-by-side comparison of the two techniques instead of comparing their stable approach data to the published outcomes of transient-based methodologies. This point is discussed in detail by Reviewer 2.

Response: We agree it is better to perform a direct, side-by-side comparison between the T7 transient protocol and the rRNA-P stable protocol. Ideally, the direct side by side comparison should be carried out by a third-party lab to avoid bias since our lab developed the rRNA-P stable protocol. Nevertheless, the T7 transient protocol studies (20-22) were performed from well-established Leishmania research labs with a minimum three or more attempts for each of the genes outlined in Table 1. Consequently, we do not believe the outcome would be different if these analyses were repeated in our lab. See also further explanations in responses to Reviewer 2 below.

Editor Comment: In line with concerns raised by Reviewer 2, the authors conclude that “dying and dead cells were caused by the disruption of all wild type gene allele present in these clones”, however presumably due to the obvious difficulties inherent with this, they do not provide direct supportive evidence.

Response: We understand this concern. We have attempted to extract the genomic DNA from those dying and dead Leishmania cells in the wells of 96 well plates. However, due to the extreme low yield and poor quality (degraded DNA), we were not able to obtain PCR products. We reasonably believe the disruption of all wild type essential gene alleles in these clones is the most likely cause of the cell dying and death. To better explain and illustrate how gene essentiality is normally determined with the rRNA-P stable protocol, we have now rewritten the section of essential genes (line 170-232) using the AGC essential kinase 1 gene (LmxM.25.2340, AEK1) as a typical example (see new Fig. 2).

See also response below to reviewer 2 that further explains the interpretation of dead and dying clones.

Different from the T7 transient protocol, the gRNA and Cas9 are constantly expressed in the stable CRISPR protocol. Once the CRISPR plasmid pLdCN (pLdsaCN) was transfected into Leishmania promastigotes, the gRNA/Cas9 complex would continue scanning the genome until all the WT alleles were targeted and disrupted. If the gene is required for viability and once the remaining copy of the essential gene has been disrupted by CRISPR, cells in those wells would stop growing or multiply slowly to form clumps until the gene products (mRNA and protein) are diluted and degraded to the minimum level required for survival. Depending on the relative importance, initial abundance, and stability of the gene product in the cell and the cloning time (stage) post the complete gene disruption for the individual clone, the dying (dead) cell clumps could contain only a few cells to more than hundreds of cells (new Fig. 2A and see below). (Note: if the cell dying was caused by the failed double strand DNA break repair, the promastigote would stop proliferating right after cloning and would not be able to form clumps containing more than ten to hundreds of cells; if the cell dying was caused by a spontaneous rare lethal mutation in the genome (unlikely), much fewer dying clones (maybe only one clone if it did happen) would be observed in a 96 well plate, instead of more than three dying clones regularly observed for disruption of an essential gene). PCR analysis will show the WT gene band persists in all surviving clones and at least one allele of the essential gene was successfully targeted and disrupted by CRISPR (new Fig. 2B). In this manner by combining observation of death of gene null mutant clones and detection of the WT gene band and the gene targeting band in all surviving clones, using rRNA-P stable protocol, we were able to confirm with confidence that many of those genes which T7 system was not able to generate alive null mutants (14 out of 22, see table 1, new Fig 2, new Fig 3 and S1 Fig ) are truly essential for Leishmania viability.

Editor Comment: The LmxM.20.1180 null mutant was described in the text as normal in mobility, yet the swimming assay data in Fig 5B indicate that it is more than twice as mobile as WT. This should be accounted for.

Response: We agree that a more precise description should be added to the text regarding LmxM.20.1180 null mutant. We have now revised the sentence (line 297-299) as below:

In comparison, the LmxM.20.1180 (CALP1.1) null mutants are normal in size but appeared to be able to proliferate and swim slightly faster than the wild type cells (S5 movie and Figs 5 and 6).

Editor Comment: In Fig. 2D, please account for the additional fainter band running at 700 bp in the Cal (+--/---) lane.

Response: For simplicity we did not include the detailed PCR analysis data in the old Figure 2 in the version submitted. We have now rewritten the text regarding the Calmodulin gene targeting and included the detailed PCR analysis data on the Calmodulin (+--/---) clone with additional PCR primers in the new Figure (now Figure 3) (line197-232). As you can see in Fig. 3, PCR analysis revealed that CRISPR gene targeting had deleted both alleles of LmxM 09.0930, disrupted both alleles of LmxM 09.0910 and one allele of LmxM 09.0920, and only one wild type LmxM 09.0920 allele remained in this slowest growing clone (Fig. 3B&C). It is interesting to note: a smaller than the expected size (R+10R1) band was detected in this Cal (+--/---) clone, indicating a recombination deletion had occurred in one of the two disrupted LmxM 09.0910 alleles, which also explains the additional fainter band running at 700 bp detected in the PCR with (R+L) primer pair.

Editor Comment: Many minor issues with language throughout the manuscript that need to be edited.

Response: We have carefully gone through the manuscript and made corrections if necessary.

Editor Comment: In addition to these points, please also address all points raised by Reviewer 2.

Response: We have responded to all concerns raised by Reviewer 2.

Reviewer 1

Comment: This is a timely and very important paper that provides important information on one of the most used techniques: gene deletion. Moreover, we, the scientists in the field of leishmaniasis, struggle to be sure about the essentiality of the gene we delete from the parasite genome. To date, CHRISPR is the most used technology for gene deletion. This study provides an important and useful guide on this topic. The paper is well written and describes a carefully designed study. Hence, this paper should be accepted for publication as is.

Response: We thank Reviewer 1 for his understanding and support of this study.

Reviewer 2

Comment: This paper reports the results of gene deletion attempts with a specific CRISPR protocol (named rRNA-P “stable” protocol). 22 genes were targeted, which in previous studies that used a different CRISPR protocol (“transient” protocol) did not yield confirmed null mutants. The authors show that they successfully knocked out some of these genes, and for some they also failed to obtain null mutants.

Response: We agree.

Comment: The stated aim, broadly, is to determine whether the “stable” protocol could be used to “define genes as essential in Leishmania”. The authors conclude that “This study demonstrates the advantage of performing the rRNA stable protocol to confirm gene essentiality […].” (line 128) This conclusion is flawed. Fundamentally, failure to generate a viable knock-out is never sufficient to prove that said gene is “essential”. Jones et al. 2018 (PMID: 29384366) have provided the most comprehensive analysis to date of reverse genetically engineered knockout mutants in Leishmania. They rehearsed in detail how a combination of different genetic approaches can increase the confidence with which a claim of “essentiality” can be made. That landmark study was not cited, but these considerations are highly relevant.

Response: The literature review article by Jones et al. 2018 (PMID: 29384366) proposes a 5-star method for determining gene essentiality which we generally agree with and have now included this reference in the revised paper (ref 2). In practice however, it is not practical to meet the 4–5-star stringent criteria proposed by Jones et al using forced plasmid shuffling and DiCre gene deletion methods which are labor intensive and time consuming, for which there are only a few examples in the literature for Leishmania. Actually, the rRNA-P stable CRISPR is similar to the DiCre inducible gene deletion method as both methods rely on observation of cell death after complete disruption or deletion of the essential genes.

Because of its simplicity and effectiveness, CRISPR has revolutionized gene targeting methods in almost all living organisms. We believe it is also time to embrace the new CRISPR technology for determining gene essentiality in Leishmania. For example, Dr. J.C. Mottram group, the authors of the above review article, has set up a new diagnosis standard for essential genes with the T7 CRISPR transient protocol; that is a gene can be considered essential if the alive null mutant could not be generated after minimum three attempts of the T7 transient method (see nature communication (reference 21) “If a gene deletion mutant was unable to be identified after three transfection attempts, it was classed as ‘required’ with a 1-star quality classification. Forty-three protein kinases and AMPKγ fell into this category”) [21]. As verified by our stable CRISPR protocol in this study, three attempts’ method did correctly predict the gene essentiality for 10 of the 12 kinase genes which the T7 transient protocol failed to isolate the alive null mutants after three attempts in reference 21 study. If the PKAC1 and PKAC2 are excluded because failure to generate those null mutants were likely due to the primer design error in the references 20 and 21 studies, the predication accuracy is 100% (10/10). However, verified by this study, this three attempts’ method has only 43% (3/7) accurate rate in predicting the gene essentiality for 7 L. donovani genes from reference 22 study. Therefore, we believe it is important to understand the advantages and weaknesses of the T7 transient method and the rRNA-P stable method in targeting Leishmania genes and determining their essentiality. As demonstrated in this study, (the exact statement in line 128) “These results suggest that the rRNA-P stable protocol is a useful complement to the T7 transient system for investigating gene essentiality and may be more suitable for targeting multicopy genes.”

This mentioned reference (below) has now been cited as reference 2

Jones, N. G., Catta-Preta, C. M. C., Lima, A. P. C. A. & Mottram, J. C. Genetically validated drug targets in Leishmania: current knowledge and future prospects. ACS Infect. Dis. 4, 467–477 (2018).

Consistent with the Jones review article (ref 2) we have changed the title of the paper to: “Evidence for gene essentiality in Leishmania using CRISPR”

Regardless of how essential genes are classified in Leishmania, this paper highlights one important consideration. As described, the rRNA stable protocol should be considered to generate the null mutant when it is not possible to generate a null mutant with the T7 transient protocol. The ability to generate null mutants is essential for studying differentiation, virulence, pathogenesis and basic biology of the parasite. This is now highlighted in the discussion of the revised version on lines 338-342.

Comment: The attempts to compare the “stable” and the “transient” methods are flawed too. Using the two methods in parallel to target the same genes and compare results would allow for a comparison. Instead, results were taken from studies with different goals (generating large numbers of KOs, prioritising throughput) and compared here against the goal of isolating null mutants for a small handful of genes. This is a comparison of two very different experimental workflows from which few conclusions can be drawn about the relative power of these methods to achieve gene knockouts. The key difference is clonal vs. population analysis, rather than “stable” vs. “transient” CRISPR methods.

Response: As our response to editor, to avoid preference, ideally, comparison between the stable and transient CRISPR methods should be performed in parallel to target the same batch of genes by a third-party laboratory.

We understand generating hundreds of Leishmania gene Knockouts with the T7 transient method were challenging in the previous three studies (see reference 20,21and 22). However, it is not true that less effort was put to isolate the individual gene null mutants in those three high throughput studies. As mentioned above, a minimum of three transfection attempts of T7 transient protocol were performed in order to class a gene as essential according to the three attempts diagnosis standard (see reference 21). In fact, 5 transfections were attempted for the non-essential L. donovani genes LdBPK_100590 and LdBPK_230540 but the T7 transient method somehow failed to generate the null mutants. Furthermore, for unknown reasons, the T7 transient method was still not able to generate the chromosome alleles deletion mutant for LdBPK_100590 gene even after an episomal copy of the gene was provided to the parasite (see Fig. 2 in reference 22).

For LmxM.34.4010 gene (PKAC1), likely because both the authors of reference 20 and 21 (two of the best Leishmania research labs in the world) did not realize that PKAC1 and PKAC2 (LmxM.34.3960) possess a large identical sequence at 3’ end (see S2 Fig), the primers used to verify the gene deletion in reference 20 and 21 were from the sequence shared by both PKAC1 and PKAC2. Therefore, detection of the WT gene band after targeting with the T7 system could be due to the non-specific primers used for PCR analysis for those genes (see reference 21). Regrettably, detection of the gene band shared by PKAC1 and PKAC2 after targeting PKAC1 gene with the T7 transient method has been used as an example how an essential gene was determined by PCR analysis (see Fig.2b in reference 21). A similar mistake was made in targeting LmxM.20.1180 gene in reference 20 study (see S3 Fig).

It could be true that if cloning was applied, the null mutants could have been isolated by the T7 transient protocol for some of the five non-essential genes identified by the rRNA-P stable system in this study. However, because two antibiotic repair donors were typically used in the T7 transient method, indeed, null mutants for many non-essential genes could be obtained without cloning if these non-essential genes are present in diploid chromosomes and their deletions have no inhibition effect on the parasite growth such as most of the flagellar protein genes, this has given the impression to Leishmania research community that no cell cloning is needed when using T7 transient CRISPR method other than repeating transfections. Therefore, our advice is that one can continue using T7 transient method without cloning for the first attempt. However, if the null mutant cannot be obtained after the first try, it might be more efficient to clone the transfectants following the second transfection attempt rather than repeating the transfection many times without cloning. If after cloning, a null mutant still cannot be isolated by the T7 transient method, then it is time to consider the stable CRISPR expression approach.

It is important to note that this study was initiated by our curiosity why the deletion mutants for four of the Leishmania flagellar protein genes could not be generated by the T7 transient method. After blast search and sequence analysis, we realized that fai

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PLoS One. doi: 10.1371/journal.pone.0316331.r003

Decision Letter 1

Ben L Kelly

22 Oct 2024

PONE-D-24-31151R1Evidence for gene essentiality in Leishmania using CRISPRPLOS ONE

Dear Dr. Zhnag,

Please submit your revised manuscript by Dec 06 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #2: The revised manuscript addresses the main issues raised in the review of the initial submission and provides additional control experiments, which were needed to support the conclusions.

The detailed responses to the reviewers’ comments were particularly helpful in clarifying some interpretations of the data and the revised figures are easier to follow.

The claims around “proving” gene essentiality are better justified – the “stable” CRISPR protocol is (mostly) presented as a valuable tool to support claims of essentiality, rather than a tool to determine essentiality.

Importantly, there is good evidence in this paper that the “stable” CRISPR protocol is suitable to determine the KO phenotypes of multicopy genes. In my opinion this is the greatest strength of this method, which no doubt is a very useful tool for reverse genetics studies on Leishmania.

Most of the conclusions are supported by the data:

Figure 1 convincing evidence for successful KO of Ld100590 and Ld230540.

Figures 2 and 3 provide data that are consistent with essential functions for LmxM.25.2340 and calmodulin.

Figure 4. Supports the conclusion that Ld310120, Ld312380 and LmxM.16.1550 were knocked out and provides evidence that the stable protocol can be used to target genes on chromosomes with >2 copies successfully.

Figure 5. Supports the conclusion that some mutants grow more slowly as promastigotes. (The cell density [cells/ml] should be plotted on a log scale because a semi-logarithmic plot allows for the direct comparison of the growth rates during the exponential growth phase.)

Figure 6 supports the conclusion that some mutants are impaired in their motility.

There are still a few sections where clarification would be helpful (some of this information was provided in the extensive response to reviewers – here my suggestions where it would be helpful to add explanations to the manuscript text):

(1) Interpretation of dying cell clumps.

Figure 2. Microscopic observation shows cell clumps indicative of dying cells. The images support the conclusion that these clonal cell lines are not viable. 2B shows PCR products indicating the presence of the wild type LmxM.25.2340 gene as well as the modified gene locus. The interpretation is unclear. If the wild type gene is still detected, these are not KOs. The authors should clarify their interpretation of the PCR results and amend the figure legend as needed. Specifically:

- Did all clones die and form clumps as shown in 2A? Or did some wells still contain live cells that looked different from the clumps?

- Was the PCR in 2B done from wells with clumps (at what time point), or from wells with live cells?

- Do they conclude that one allele is insufficient for survival? Or did they conclude that cells that manage to survive until the final allele is removed. (But for technical reasons DNA can only be analyzed from cells that have not yet reached that final stage).?

There are several other sections where the assumption is made that cells were null mutants but the actual status of the gene locus could/was not determined:

line 45 (and 138-140) – “by directly observing gene null mutant promastigotes dying in culture”. The observation is: dying promastigotes. When these genes are targeted with the transient protocol, dying promastigotes are also observed. Whether or not the targeted gene is absent in these promastigotes is not known, in either case. All that can be concluded (in both protocols) is that failure to recover live cells is consistent with an essential function of the targeted gene (but not definitive proof).

Figure S1. line 647-648. “Morphology of clumping dying clones (-/-) once the remaining copy of the gene has been disrupted by CRISPR.” The PCR shows that the WT band was present in all samples. No evidence is presented to show that the remaining copy of the gene has been disrupted. It is possible that this is what happens, likely even in many cases, but the data does not establish a direct link between the loss of the gene and the death of the cells. If the authors’ interpretation was correct, there should be a decrease of the WT PCR product over time. There is no evidence that the WT band decreases with time.

The time point at which the DNA was tested should be stated.

Figure S1. line 647-648. These data do not provide evidence that the gene is essential. Other explanations for the death of the cells cannot be excluded.

For all of these, the only claim that can be made is that the observed decay of the cells is consistent with an essential function of the gene.

Note, Figure S1 legend. Lines 650-653 are a duplication of lines 224-227 in the main text.

(2) Ability to delete protein kinase A

Table 1. Null mutants for LmxM.34.3960 and LmxM.34.4010 (Protein kinase A catalytic

subunit isoforms) were not confirmed in the cited reference but they were subsequently reported in Fochler et al., Biorxiv 2023 (for completeness this could be added to footnote 6). This successful KO also used the transient protocol; it is likely it was able to confirm the KO because gene-specific primers were used, unlike the reference cited in the manuscript.

(3) Claims about calmodulin

Figure 3B, C, line 253-254. I do not follow the argument that “only one wild type LmxM.09.0920 allele remained”. There are 3 PCR bands in the mutant – it would be helpful to label them in the illustration (WT, modified locus, ...?).

How was allele copy number determined?

Line 250 “data not shown” – please show data or remove statement.

Line 257 “Calmodulin is only one of the 98 Leishmania flagellar protein genes” required for viability. It isn't clear if this means “only one of many” or “the only one”.

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Reviewer #2: No

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PLoS One. 2024 Dec 30;19(12):e0316331. doi: 10.1371/journal.pone.0316331.r004

Author response to Decision Letter 1

2 Dec 2024

Response to New additional Comments by Reviewer2

Reviewer Comment: Figure 5. Supports the conclusion that some mutants grow more slowly as promastigotes. (The cell density [cells/ml] should be plotted on a log scale because a semi-logarithmic plot allows for the direct comparison of the growth rates during the exponential growth phase.)

Response: As we explained in the previous Response to reviewers, depending on the researcher’s preference, we understand that Leishmania growth curves can also be plotted on a semi-log plot. However, like most Leishmania research labs, we prefer to use the regular plot (the actual cell density; the number of promastigotes /ml) to plot the growth curve. As shown in the supplementary PowerPoint slide (for review purpose only) of comparison between the regular plot and the semi-log plot, it clearly shows that the regular plot can more precisely display the Leishmania growth rate and is easier to distinguish the differences between cell lines.

Reviewer Comment: There are still a few sections where clarification would be helpful (some of this information was provided in the extensive response to reviewers – here my suggestions where it would be helpful to add explanations to the manuscript text):

(1) Interpretation of dying cell clumps.

- Did all clones die and form clumps as shown in 2A? Or did some wells still contain live cells that looked different from the clumps?

- Was the PCR in 2B done from wells with clumps (at what time point), or from wells with live cells?

There are several other sections where the assumption is made that cells were null mutants but the actual status of the gene locus could/was not determined:

Figure S1. line 647-648. These data do not provide evidence that the gene is essential. Other explanations for the death of the cells cannot be excluded.

For all of these, the only claim that can be made is that the observed decay of the cells is consistent with an essential function of the gene.

General response to the above reviewer comments (See specific responses below): We believe we have explained and answered the similar concerns in the previous Response to reviewers. We will explain here in detail once more, which will also answer some of the specific questions. Different from the T7 transient protocol, the gRNA and Cas9 are constantly expressed in the stable CRISPR protocol. Once the CRISPR plasmid pLdCN (pLdsaCN) was transfected into Leishmania promastigotes, followed by the antibiotic donor transfection, the gRNA/Cas9 complex would continue scanning the genome until all the WT alleles were targeted and disrupted. If the gene to be targeted is non-essential for viability, the cloned cells would continue to proliferate. Depending on the gene and/or gRNA, PCR analysis of those alive clones will show that either one gene allele or both gene alleles have been disrupted with the antibiotic selection marker, and the smaller WT gene band will not be detected in many of those clones (Fig. 1B). However, if the gene is required for viability and once the remaining copy of the essential gene has been disrupted by CRISPR, cells in those wells would stop growing or multiply slowly to form clumps until the gene products (mRNA and protein) are diluted and degraded to the minimum level required for survival. Depending on the relative importance, initial abundance, and stability of the gene product in the cell and the cloning time (stage) post the complete gene disruption for the individual clone, the dying (dead) cell clumps could contain only a few cells to more than hundreds of cells (Fig. 2A and S1 Fig). Three to ten dying null mutant clones will usually be observed in a 96 well plate when targeting an essential gene. PCR analysis of the remaining surviving clones in the 96 well plate will show the WT gene band persists in all surviving clones and at least one allele of the essential gene was successfully targeted and disrupted by CRISPR (Fig. 2B). In this manner, by combining observation of the death of gene null mutant clones and detection of the WT gene band and the gene targeting band in all surviving clones, using rRNA-P stable CRISPR protocol, we were able to confirm that many of those genes for which the T7 transient protocol was not able to generate alive null mutants (14 out of 22, see table 1, Fig 2, Fig 3 and S1 Fig) are truly essential for Leishmania viability.

As suggested by the reviewer, we have revised and added the following sentences into the manuscript text to better explain how the gene essentiality is normally determined with the rRNA-P stable CRISPR protocol:

Line208-210: “To illustrate how gene essentiality is normally determined with the rRNA-P stable protocol, we use targeting AGC essential kinase 1 gene (LmxM.25.2340, AEK1) as an example (Fig 2).”

Line228-233: “In this manner, by combining observation of the death of gene null mutant clones and detection of the WT gene band and the gene targeting band in all surviving clones, using the rRNA-P stable CRISPR protocol, we were able to confirm that many of those genes for which the T7 transient protocol was not able to generate alive null mutants (14 out of 22; see Table 1, Fig. 2, Fig. 3, and S1 Fig.) are truly essential for Leishmania viability.”

Comment: - Did all clones die and form clumps as shown in 2A? Or did some wells still contain live cells that looked different from the clumps?

Response: No, not all clones in the 96 well plate form clumps and eventually die as shown in Fig 2A; most wells in the 96 well plate still contain live cells that looked different from the clumps. These live cell wells are the wells that were used to generate the PCR results in Fig. 2B.

Although the gRNA and Cas9 are constantly expressed in the stable rRNA-P protocol, CRISPR gene targeting is still not very efficient in Leishmania. As described above, depending on the gene and the gRNA activity, 3 to 10 wells with dying null mutant clones(-/-) will usually be observed in a 96 well plate after the double antibiotic (one from pLdCN CRISPR vector, other from the antibiotic resistance repair donor) resistance promastigotes are cloned when targeting an essential Leishmania gene (Fig 2A, Fig 3D and S1 Fig). The live promastigotes (+/-) in the remaining wells will continue proliferation with various growth rate as CRISPR is still targeting the remaining essential gene allele in some of the cells to yield sufficient cells for subsequent PCR analysis. PCR analysis of those surviving clones in the 96 well plate will show the WT gene band persists and at least one allele of the essential gene was successfully disrupted by CRISPR (Fig. 2B and S1 Fig).

Comment: Was the PCR in 2B done from wells with clumps (at what time point), or from wells with live cells?

Response: The PCR in Fig. 2B (in all PCR analysis in this manuscript) was performed from wells with live cells, which is clearly stated in the text (line 226-228): “PCR analysis of all the surviving clones in the 96 well plate will show the WT gene band persists and at least one allele of the essential gene was successfully targeted and disrupted by CRISPR (Fig 2B, S1 Fig and below)”.

Comment: Do they conclude that one allele is insufficient for survival? Or did they conclude that cells that manage to survive until the final allele is removed. (But for technical reasons DNA can only be analyzed from cells that have not yet reached that final stage).?

Response: As described above and demonstrated in this manuscript, we conclude that one essential gene allele is sufficient for Leishmania parasite survival.

Comment: There are several other sections where the assumption is made that cells were null mutants but the actual status of the gene locus could/was not determined:

Figure S1. line 647-648. These data do not provide evidence that the gene is essential. Other explanations for the death of the cells cannot be excluded.

For all of these, the only claim that can be made is that the observed decay of the cells is consistent with an essential function of the gene.

Response: As we explained in our previous Response to reviewers, we had difficulty extracting the genomic DNA from the dying cell clumps for PCR analysis. However, we believe the disruption of all wild type essential gene alleles in those clones is the most likely cause of the cell dying and death as explained and illustrated in the section of essential genes (line 206-282) in the text using the AGC essential kinase 1 gene (LmxM.25.2340, AEK1) as a typical example (Fig. 2) to show how gene essentiality is normally determined with the rRNA-P stable protocol.

In addition, if the cell dying after cloning the double antibiotic resistance promastigotes into a 96 well plate was caused by the failed double strand DNA break repair, the promastigote would stop proliferating right after cloning and would not be able to form clumps containing more than ten to hundreds of cells; if the cell dying was caused by a spontaneous rare lethal mutation in the genome (unlikely), much fewer dying clones (maybe only one clone if it did happen) would be observed in a 96 well plate, instead of more than three dying clones regularly observed for disruption of an essential gene.

We understand the concern raised by this reviewer that despite the complete disruption of the essential gene alleles by the stable CRISPR gene targeting protocol is the most likely cause of cell death observed in the 96 well plate, due to difficulty to extract genomic DNA from the dying cell clumps, we did not have direct evidence so far to show that both the essential gene alleles in those dying promastigotes clumps were indeed disrupted by CRISPR with the antibiotic selection marker donor. However, it is important to point out that the DiCre inducibe gene deletion method, the most stringent method before CRISPR and recommended by this reviewer (see reference 2), also relies on observation of the cell death after complete deletion of the essential gene alleles to determine Leishmania gene essentiality. DiCre method also did not provide direct evidence (or definitive proof) to show that the floxed remaining essential gene allele in those dying Leishmania cells was indeed excised by the DiCre after addition of rapamycin.

To identify potential new drug targets, it is necessary to clearly determine the essentiality for all Leishmania genes [2]. The T7 transient system currently employs two methods to determine whether a gene is essential for Leishmania viability. Method 1 is to see, like the traditional gene targeting, if it is possible to delete the two chromosomal copies of the essential gene of interest by providing the cell with an extrachromosomal copy of the gene [2, 21,22]; Method 2 is to repeat the targeting attempt for a minimum of three times, if the null gene mutant could still not be generated with T7 system after three attempts, the gene can be considered as essential for Leishmania [21].Thus, both methods are labor intensive and time consuming. Method 2 at times could be not reliable as 4 to 6 attempts with T7 system were sometimes required to generate the null mutants for non-essential genes as shown in the L. donovani membrane protein gene study [22]. Nevertheless, as demonstrated in this study, using the rRNA-P stable protocol with single CRISPR plasmid and single antibiotic selection marker donor transfection, it was possible to determine gene essentiality by combining observation of the death of gene null mutant clones and detection of the WT essential gene band and the gene targeting band in all surviving clones in a 96 well plate [9,10,12,14].

Comment: line 45 (and 138-140) – “by directly observing gene null mutant promastigotes dying in culture”. The observation is: dying promastigotes. When these genes are targeted with the transient protocol, dying promastigotes are also observed. Whether or not the targeted gene is absent in these promastigotes is not known, in either case. All that can be concluded (in both protocols) is that failure to recover live cells is consistent with an essential function of the targeted gene (but not definitive proof).

Response: Although the complete sentence in line 43 should be “The rRNA-P stable protocol provides evidence for gene essentiality by directly observing null mutant promastigotes dying after cloning the antibiotic resistance promastigotes into a 96 well plate”, we believe line 43 (and 127-128) – “by directly observing gene null mutant promastigotes dying in culture” is appropriate in Abstract and Introduction because the Results section text will explain what it means the dying null promastigotes in culture after cloning into a 96 well plate.

As we explained in our previous Response to reviewers, the dying promastigotes observed in transient protocol could be caused by several reasons (1) the non-transfected wild type cells killed by the two selecting antibiotics; (2) the cells killed by the two selecting antibiotics which were successfully transfected but no gene targeting took place; (3) the partially targeted (only one allele was targeted) cells killed by the second selecting antibiotic; (4) the cells with both alleles targeted by the same antibiotic selection marker donor, those cells would be killed by the another selecting antibiotic; (5) the cells with both alleles successfully targeted by each of the two antibiotic selection marker donors, dying and death of those cells after adding the two selecting antibiotics would be caused by the deletion of the targeting gene if it is essential. Thus, it is very difficult in the transient protocol to distinguish the cause of cell death between failure of the gene targeting and essentiality of a gene in such a large dying cell population.

In contrast, in the stable protocol, only one antibiotic selection marker donor is provided to the parasite. Before cloning into a 96 well plate, the promastigotes are already selected by the antibiotic specific to the selection marker donor, only cells with one or both alleles correctly targeted survive (if the gene is essential, the cells with both alleles disrupted will eventually die, see below). Because the stably expressed Cas9 and gRNA will continue to scan the genome and target the remaining WT gene allele after cloning into a 96 well plate, if the gene is essential for Leishmania viability, once the remaining WT allele in a cloned promastigote is targeted and disrupted, promastigotes in those wells would stop growing or multiply slowly to form clumps until the gene products (mRNA and protein) are diluted and degraded to the minimum level required for survival. Therefore, the slow dying of the cloned cells in the stable protocol could only result from the complete disruption of the essential gene alleles. In comparison, as described above, multiple factors could cause cell death in the transient protocol.

Comment: Figure S1. line 647-648. “Morphology of clumping dying clones (-/-) once the remaining copy of the gene has been disrupted by CRISPR.” The PCR shows that the WT band was present in all samples. No evidence is presented to show that the remaining copy of the gene has been disrupted. It is possible that this is what happens, likely even in many cases, but the data does not establish a direct link between the loss of the gene and the death of the cells. If the authors’ interpretation was correct, there should be a decrease of the WT PCR product over time. There is no evidence that the WT band decreases with time.

Respo

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PLoS One. doi: 10.1371/journal.pone.0316331.r005

Decision Letter 2

Ben L Kelly

11 Dec 2024

Evidence for gene essentiality in Leishmania using CRISPR

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PLoS One. doi: 10.1371/journal.pone.0316331.r006

Acceptance letter

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13 Dec 2024

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Evidence for Leishmania essential genes included in Table 1 using the rRNA-P stable CRISPR protocol.

(PDF)

pone.0316331.s001.pdf^{(1.3MB, pdf)}

S2 Fig. Successful gene disruption of non-essential L. mexicana PKAC1 (LmxM.34.4010) and PKAC2 (LmxM.34.3960) with rRNA-P stable protocol.

(PDF)

pone.0316331.s002.pdf^{(68.6KB, pdf)}

S3 Fig. Successful gene disruption of L. mexicana CALP1 (LmxM.20.1180) with rRNA-P stable protocol.

(PDF)

pone.0316331.s003.pdf^{(51.6KB, pdf)}

S1 Table. Oligonucleotides and primers used in this study.

(DOCX)

pone.0316331.s004.docx^{(29.9KB, docx)}

S1 Movie. Leishmania mexicana wildtype promastigotes in culture.

(MOV)

pone.0316331.s005.MOV^{(10.8MB, MOV)}

S2 Movie. LmxM.16.1550 (CMF6) null mutant promastigotes in culture.

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S3 Movie. LmxM.34.4010 (PKAC1) null mutant promastigotes in culture.

(MOV)

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S4 Movie. LmxM.09.0910 (Calmodulin) partial deletion mutant (+—/—) promastigotes in culture.

(MOV)

pone.0316331.s008.MOV^{(11MB, MOV)}

S5 Movie. LmxM.20.1180 (CALP1.1) null mutant promastigotes in culture.

(MOV)

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S1 Raw images

(PDF)

pone.0316331.s010.pdf^{(480.1KB, pdf)}

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Evidence for gene essentiality in Leishmania using CRISPR

Wen-Wei Zhang

Greg Matlashewski

Roles

Abstract

Introduction

Results

Non-essential genes

Table 1. Comparison of two CRISPR methods in targeting Leishmania genes.

Fig 1. CRISPR gene targeting strategy (rRNA-P stable protocol) used to disrupt the essential and non-essential Leishmania genes in this study.

Essential genes

Fig 2. Evidence the AGC essential kinase 1 gene (LmxM.25.2340, AEK1) is essential for L. mexicana.

Fig 3. Evidence the Calmodulin gene is essential for L. mexicana.

Genes on polyploid chromosomes

Fig 4. Disruption of non-essential Leishmania genes in polyploid chromosomes with rRNAP-P stable protocol.

Genes required for optimum proliferation

Fig 5. Promastigote growth curves of the L. donovani and L. mexicana null mutants generated in this study.

Phenotypic changes in gene disrupted mutants

Fig 6. Mobility defects were observed in L. mexicana null mutants generated in this study.

Discussion

Materials and methods

Leishmania strains and culture medium

gRNA design and cloning

Parasite transfection

Cloning into 96-well plates

Genomic DNA preparation and PCR analysis

Growth curves and cell imaging

Promastigote motility assay

Supporting information

Data Availability

Funding Statement

References

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Ben L Kelly

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Author response to Decision Letter 0

Decision Letter 1

Ben L Kelly

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Ben L Kelly

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Acceptance letter

Ben L Kelly

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Associated Data

Supplementary Materials

Data Availability Statement

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