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. Author manuscript; available in PMC: 2018 Jun 15.
Published in final edited form as: Dev Biol. 2016 Jun 16;426(2):261–269. doi: 10.1016/j.ydbio.2016.05.023

Exploring the functions of nonclassical MHC genes in Xenopus laevis by the CRISPR/Cas9 system

Banach Maureen 1, Edholm Eva-Stina 1, Robert Jacques 1
PMCID: PMC5501940  NIHMSID: NIHMS876443  PMID: 27318386

Abstract

A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X laevis.

Keywords: Gene knockout, reverse genetic, genomics, amphibians, comparative immunology, XNC

Introduction

In jawed vertebrates, adaptive immunity relays on a set of genes known as major histocompatibility complex (MHC) that encode antigen binding cell surface molecules. By displaying a specific antigen on MHC molecules, a cell informs the immune system whether it is healthy, infected, or cancerous. In the thymus, MHC molecules are also required for the education of developing T cells. The MHC gene family is categorized into two main groups: MHC class I and class II, with MHC class I genes further subdivided into classical MHC class Ia (class Ia) and nonclassical MHC class Ib (class Ib) genes. The primary structure of both class Ia and class Ib molecules consists of three alpha domains (α1, α2, α3), a transmembrane region, and a cytoplasmic tail. The α1 and α2 domains form an antigen binding pocket, whereas the α3 domain typically associates with beta2 microglobulin (Adams and Luoma, 2013; Bjorkman et al., 1987).

The significance of class Ia for the development, education, and function of cytotoxic CD8 T cells is well established (Murphy, 2008). In contrast, class Ib is a heterogeneous group of molecules, some of which bind peptides, others lipids, glycolipids or vitamin derivatives (Kjer-Nielsen et al., 2012; Koch et al., 2005; Rossjohn et al., 2012; Van Rhijn et al., 2015). Furthermore, class Ib molecules can interact with a broader range of immune cells, including NK, γδ T, and innate T (iT) cells to which belong natural killer T (NKT) and mucosal-associated invariant T (MAIT) cells (Macho-Fernandez and Brigl, 2015). In contrast to a few highly polymorphic class Ia genes, class Ib molecules are typically encoded by many oligomorphic genes. From an evolutionary perspective class Ia gene homologs are well preserved across all vertebrates (reviewed in (Flajnik and Kasahara, 2001)). Conversely, the relationships between evolutionarily distant class Ib genes are more difficult to establish, and only a few class Ib orthologs such as the CD1 family and MR1 have been identified (Dascher, 2007; Huang et al., 2009). However, MR1 is conserved only within mammals, whereas CD1 is found in mammals, birds, and reptiles but in neither fish nor amphibians (Brossay et al., 1998; Dascher, 2007; Miller et al., 2005; Riegert et al., 1998; Salomonsen et al., 2005; Tsukamoto et al., 2013; Yang et al., 2015). Despite the lack of orthology, the genomes of ectothermic vertebrates, including urodele amphibians, Xenopus, cartilaginous, and teleost fish harbor extensive families of distinct class Ib genes (Bartl et al., 1997; Dijkstra et al., 2007; Edholm et al., 2013; Flajnik et al., 1993; Goyos et al., 2011; Sammut et al., 1999; Star et al., 2011).

Due to their critical regulatory roles in immunity, class Ib and interacting iT cells such as iNKT and MAIT cells have emerged as suitable therapeutic targets against viral, microbial, and parasitic infections, autoimmune and allergic diseases, as well as malignant neoplastic growths (Fernandez et al., 2014; Juno et al., 2012; Macho-Fernandez and Brigl, 2015; McEwen-Smith et al., 2015; Robertson et al., 2014; Skold and Behar, 2003; Subleski et al., 2011). However, the clinical implementation of class Ib- and iT cell-based therapies is still far from realization and requires a deeper and more comprehensive understanding of the biology of this a system.

In this regard, the X. laevis tadpole provides a valuable alternative model for investigating the roles of class Ib molecules and iT cells. Overall, the immune system between X. laevis and mammals is well conserved (Robert and Ohta, 2009). However, unlike mammals in which class Ia deficiency is often lethal, X. laevis tadpoles remain immunocompetent despite the lack of class Ia protein expression (Flajnik et al., 1986). Interestingly, X. laevis nonclassical MHC class Ib (XNC) genes are evolutionary conserved to their counterparts found in X. tropicalis both in the genomic organization and phylogenetic relationships (Edholm et al., 2014). Since the strong gene selection maintained for over 65 million years of evolutionary history implies important biological functions; we postulated that in tadpoles class Ib genes compensate class Ia deficiency (Evans, 2008; Robert and Edholm, 2014). In support of this hypothesis, using deep sequence analysis, we found evidence of six dominant unique iT cell subsets in X. laevis tadpoles, which could be restricted by some of these XNC molecules (Edholm et al., 2013).

A notable XNC gene, XNC10.1, is highly conserved among 12 species of the Xenopodinae subfamily (Edholm et al., 2014). Furthermore, XNC10.1 encodes a molecule required for the development and function of an iT cell population expressing an invariant T cell receptor rearrangement Vα6-Jα1.43 (Edholm et al., 2013). Loss-of-function combining RNA interference and transgenesis in X. laevis has revealed that Vα6-Jα1.43 iT cells are involved in antiviral and anti-tumoral immunity (Edholm et al., 2013; Edholm et al., 2015; Haynes-Gilmore et al., 2014). Apart from XNC10.1 transcripts, we have identified several other XNCs including XNC1 and XNC11 that are preferentially expressed by immature thymocytes, a hallmark of class Ib-mediated iT cell differentiation in mammals. As such, XNC1 and XNC11 genes could serve as restricting elements for the development of different iT cell populations in Xenopus. However, to determine the requirement of any of these XNCs for the development of putative iT cell subsets, a convenient and reliable reverse genetics loss-of-function is needed.

Although we have successfully used RNA interference (RNAi), the recent advance in genome editing technology using the CRISPR/Cas9 system is attractive (Blitz et al., 2013; Harrison et al., 2014; Nakayama et al., 2013). In this technique, a complex of Cas9 enzyme and a single stranded RNA (sgRNA) recognizes a specific genomic site and induces a double strand DNA break. An imprecise DNA repair of this break introduces mutations such as deletions or insertions that subsequently eliminate the gene product. Besides the ease in designing sgRNA targeting virtually any site in the genome, the CRISPR/Cas9 system has the advantage over the RNAi approach as it can completely and stably disrupt the gene product and function. However, the use of this technique in X. laevis still awaits more validation from specific functional studies, especially with regard to immunity (Wang et al., 2015). In addition, the XNCs locus imposes the challenge of a large family of linked genes clustered in a small genomic segment of 700 kb and sharing a relatively high level of sequence similarity. The nucleotide sequence similarity among XNCs ranges from 48 to 95% for α1; 54 to 95% for α2 and 74 to 99% for α3 domains (Edholm et al., 2014). Moreover, X. laevis has undergone allotetraploidization during which two genomes from separate species were combined, giving rise to cytologically distinguishable long (L) and short (S) chromosome pairs (Evans, 2007; Matsuda et al., 2015). The X. laevis genome assembly version 9.1 includes the attribution of gene models to L and S chromosomes (Session et al. submitted). Interestingly, based on analysis of this genome assembly the majority of XNC genes are clustered on chromosome 8L, whereas only a few XNCs reside on chromosome 8S in a region that is not syntenic. Therefore, taking advantage of the newly available X. laevis genome assembly, we explored the feasibility and effectiveness of the CRISPR/Cas9-mediated genome editing for investigating XNC genes’ functions.

Our findings clearly establish the CRISPR/Cas9 technology as the system of choice to elucidate the functions of the XNC gene family by showing its efficiency and absence of off-target effects. In addition to confirming the requirement of XNC10.1 gene in the development of Vα6 iT cells, an unexpected essential function of XNC1 during early development has been uncovered.

Materials and Methods

Animals

Outbred and inbred J strain X. laevis were obtained from our Xenopus laevis Research Resource for Immunology at the University of Rochester (http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). All animals and embryos were handled under strict laboratory and UCAR regulations (Approval number 100577/2003-1S51), minimizing discomfort at all times.

Genomic organization of XNC gene family

Chromosome numbers and gene locations are based on the UCSC Genome Bioinformatics hosted at the Francis Crick Institute (http://genomes.crick.ac.uk/) with the new genome assembly of X. laevis J strain version 9.1 (Session et al. submitted).

Microinjections of X. laevis embryos

Outbred and J strain X. laevis females were primed with 10–20 IU and boosted with 20–40 IU of human chorionic gonadotropin (Sigma, St. Louis, MO) 1 day before egg collections. To prepare a sperm suspension, two testes from either an outbred or a J strain X. laevis male were homogenized in 2 ml of 1X De Boer's solution (100 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2 and titrated to pH 7.2). Eggs were fertilized in vitro with 0.5 ml of the prepared sperm suspension. The fertilization was carried on for 10 min. Subsequently, the embryos were dejellied for 10 min with 2% (w/v) cysteine-HCl made in 0.1X modified Barth’s saline (MBS; 10X MBS prepared with 880 mM NaCl, 10 mM KCl, 10 mM MgSO4, 50 mM HEPES, 25 mM NaHCO3, pH 7.4–7.6, and autoclaved). Next, the embryos were washed three times with 0.1X MBS and placed into 0.3X MBS containing 4% Ficoll® PM 400 (Sigma). Using standard microinjection techniques and PLI-100 Pico-injector Microinjection System (Harvard Apparatus, Holliston, MA), each embryo was injected with 10 nl mixture of the recombinant Cas9 protein (PNA Bio Inc., diluted to 2 µg/µl; approximately 8 ng of the Cas9 protein per injected embryo) and sgRNA (200 pg/nl of each sgRNA either for single or double knockout experiments) into the animal pole. Cas9 protein and sgRNA were thawed and mixed together just before injections. After microinjection all embryos were incubated at 13°C for 4 h to delay cell division and to provide extended time for mutagenesis. The embryos were then transferred in 0.3X MBS supplemented with 5 µg/mL gentamicin (Invitrogen) and reared at 18°C until hatching. Next, tadpoles were raised in dechlorinated water at room temperature. X. laevis developmental stages were determined according to (Nieuwkoop and Faber, 1967).

Design and production of sgRNAs

Single guide RNA (sgRNA) was designed against either α1 or α2 domain of a targeted XNC gene using CRISPRdirect https://crispr.dbcls.jp/ and Zifit http://zifit.partners.org/ZiFiT/ online tools. The targeted sites followed the requirement of 5’- GG-n(18–20)-NGG −3’ with NGG being a protospacer adjacent motif (PAM) sequence. Importantly, using CCTop (http://crispr.cos.uni-heidelberg.de/) we initially confirmed the absence of significant match for these sgRNAs with other genomic sequences. To obtain sgRNA for microinjections, a two-step process (PCR and in vitro transcription) was performed, according to a published method (Bassett et al., 2013; Bhattacharya et al., 2015). Briefly, three different synthetic oligonucleotides for targeting XNC10.1, XNC11 or XNC1 genes were designed and obtained from Sigma. Each of these oligonucleotide contained a T7 promoter, the identified sgRNA sequence and part of the sgRNA scaffold (5’-CTAGCTAATACGACTCACTATA-GG-n(18–20)-GTTTTAGAGCTAGAAATAGCAAG-3’). Furthermore, we obtained a common synthetic oligonucleotide encoding the rest of the sgRNA scaffold. Using overlap extension PCR with the Phusion polymerase (New England Biolabs) and without additional template, these oligos were used to generate a double stranded DNA product from which the complete sgRNAs could be in vitro transcribed. Using T7 Megashortscript kit according to the manufacturer’s instructions (Ambion), sgRNAs were transcribed, purified with phenol-chloroform-isoamyl alcohol (Invitrogen), and diluted to 200 pg/nl with molecular biology grade water (Corning, NY) and stored in aliquots at −80°C.

Semi-quantitative (RT) and Quantitative PCR Gene Expression Analysis

Total RNA and DNA were extracted from whole tadpole bodies using the TRIzol reagent (Invitrogen), according to the manufacturer directions. Using 500 ng of total DNAse-treated (Ambion, CA) RNA, cDNA synthesis was performed using the M-MLV reverse transcriptase and oligo dT (Invitrogen). One microliter of these respective synthesized cDNA samples were used as templates for reverse transcription RT-PCR and real time qPCR analysis. PCR products were resolved on 1.5% agarose gels, visualized with ethidium bromide (BioRad, Hercules, CA) and compared against a 1 kb plus DNA ladder (Invitrogen). RT-PCR parameters were as follows: 5 min at 95°C followed by 38 cycles of 95°C for 30 sec, 72°C for 40 sec, and 57°C for 1 min. Relative quantitative PCR (qPCR) gene expression analysis for Vα6-Jα1.43 transcripts was performed using the ΔΔCT method (where CT is a threshold cycle), relative to the endogenous GAPDH control and normalized against the lowest observed expression (Grayfer et al., 2015). The experiments were performed using the ABI 7300 real-time PCR system and Perfecta SYBR Green FastMix, ROX (Quanta Bioscience, Gaithersburg, MD). Expression analysis was performed using ABI Sequence Detection System software. All primers were validated prior to use.

Mutation analysis

The regions targeted by the Cas9 protein and specific sgRNA were PCR amplified with genomic primers. PCR parameters were as follows: 5 min at 95°C followed by 40 cycles of 95°C for 30 sec, 72°C for 40 sec, and 57°C for 1 min, with final 20 min extension. The PCR products were resolved on 1.5% agarose gel and visualized with ethidium bromide (BioRad). The bands were excised and purified using gel extraction kit (Qiagen, Valencia, CA), ligated into pGEM-T Easy vector (Promega, Madison, WI) and cloned. Colony PCRs were performed to confirm the presence of the PCR-amplified gene fragment. We selected 3 to 19 clones per analyzed tadpole to acquire sufficient coverage. Plasmid minipreps were submitted to ACGT, Inc. DNA Sequencing Services (Wheeling, IL), for the single pass sequencing. Sequences were analyzed using the BioEdit Sequence Alignment Editor.

Statistical analysis

Statistical significance of survival data were determined by a Log-rank (Mantel-Cox) test using the GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA)

Results

Genomic organization and chromosome attribution of XNC genes

Previously, the genomic organization of XNC genes was based on the assembly version 6.1 from November 2012, which was assigned onto scaffolds and not fully annotated (Edholm et al., 2014). However, a detailed gene assignment and genomic arrangement are required for advancing our research on the specific roles of different XNC genes. This is of particular interest since previous evidence from comparative Southern blot and genome sequence analysis between the diploid X. tropicalis and the allotetraploid X. laevis indicated that XNC gene number is reduced or diploidized (Edholm et al., 2014; Flajnik et al., 1993). We took advantage of the newly released X. laevis genome assembly version 9.1 that includes chromosome mapping to gain further insights into the genomic location and organization of the XNC locus (Xenbase.org; Session et al. submitted).

We first assessed the location of each XNC gene on long (L) and short (S) chromosome pairs. Matching with the genomic location of X. tropicalis class Ib genes (Edholm et al., 2014), the majority of XNC genes is located on X. laevis chromosome 8L and spans over 700 kb (Fig. 1A). Specifically, within this genomic segment, there are 17 XNC genes that have been validated by expression profiling using expressed sequence tags (ESTs) and/or reverse transcription RT-PCR (Edholm et al., 2014; Flajnik et al., 1993; Goyos et al., 2011). All of these XNC genes, except XNC7, have the same transcriptional orientation. Furthermore, XNC2, XNC14 genes and XNC6.4 pseudogene are located away from the major XNC locus, namely, on the chromosome 8S within a 70 kb segment. Finally, one scaffold (168) containing a family of XNC1 genes has not yet been assigned to a particular chromosomal location. Whether the location of XNC genes within or away from the major locus influences their biological functions requires further investigation.

Figure 1. Genomic organization of X. laevis nonclassical MHC (XNC) genes and the CRISPR/Cas9 targeting strategy.

Figure 1

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(A) Genomic organization of 21 XNC genes according to the version 9.1 assembly, August 2015. Seventeen XNC genes are located on chromosome 8L within a region of 700 kb (drawn to scale). XNC2, XNC14 genes and XNC6.4 pseudogene (indicated by *) are located on the chromosome 8S. Chromosome attribution of the orphan scaffold 168 containing XNC1 is still missing. Transcriptional orientation of XNC genes is indicated by arrows. Chromosome number and gene location are based on the UCSC Genome Browser hosted at Francis Crick Institute (B) Out of 21 XNC genes XNC1, XNC11, and XNC10.1 were targeted by the CRISPR/Cas9 system in order to generate knockout transgenic X. laevis. The sgRNAs were designed against α1 or α2 domain of the targeted genes. The targeted regions are underlined, follow GG-n(18-20) schema, and have a required PAM sequence. The sgRNAs were designed with the aid of the online tools (CRISPRdirect: https://crispr.dbcls.jp/ and Zifit: http://zifit.partners.org/ZiFiT/).

Strategy to generate XNC genes knockout X. laevis tadpoles by the CRISPR/Cas9 system

In previous studies with the aid of RNAi technology, we generated XNC10.1-deficient X. laevis that had reduced number of XNC10.1-restricted iT cells (Edholm et al., 2013). However, high variability in the degree and duration of gene silencing by RNAi has complicated functional studies. Thus, we thought that a complete knockout of XNC10.1, generated by the CRISPR/Cas9 system, would help overcome these obstacles. We designed a single guide RNA (sgRNA) against XNC10.1 α2 domain in order to efficiently knock out the XNC10.1 gene product. Furthermore, we previously found that both XNC11 (Goyos et al 2009) and XNC1 (unpublished observations) are highly expressed by immature thymocytes, which in mammals is a hallmark of class Ib-mediated iT cell development, and by Xenopus thymus-derived lymphoid tumor cell lines. This suggests important immune functions, possibly critical for the development and function of distinct iT cell subsets. Thus, we also generated single knockouts of XNC11 and XNC1 genes (Fig. 1B). We designed different sgRNAs using bioinformatics online tools as described in the materials and methods section.

Generation of XNC10.1 knockout X. laevis tadpoles

Out of 400 growing embryos that were injected with the complex of Cas9 protein and sgRNA against XNC10.1 α2 domain, 10 tadpoles at the developmental stage 47 (Nieuwkoop and Faber, 1967) were randomly chosen for genomic and transcriptional analysis. To uncover the mutation frequencies and potential somatic mosaicism, genomic DNA of the XNC10.1 α2 domain was amplified by PCR and sequenced with a 3 to 5 times coverage. All the sequences were aligned and compared to the wild type (WT) sequence of the XNC10.1 α2 domain (Fig. 2A). Apart from two tadpoles for which none of the sequences analyzed were mutated, we found that 8 out of 10 tested tadpoles had all XNC10.1 α2 domain sequences mutated. This overall amounted to an 80% mutagenesis efficiency. Within the combined 38 mutated sequences from all 8 tadpoles, we found that the majority of mutations (68.2 %) were out-of-frame deletions (Fig.2B). Such deletions should lead to the elimination of the functional gene product. Within individual tadpoles, a high degree of mosaicism was also observed; possibly due to the method used (see discussion). However, some deletion types occurred more than once across different animals. Notably, a 7-bp deletion was found in four different tadpoles (Fig. 2A). This preference of mutation types may indicate some inherent characteristic of the CRISPR/Cas9 technology itself, although further investigation will be needed to validate this claim.

Figure 2. XNC10.1 loss-of-function by CRISPR/Cas9-mediated editing of XNC10.1 α2 domain.

Figure 2

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(A) Multiple alignments of a portion of the XNC10.1 α2 domain targeted by the Cas9 protein and sgRNA from 8 different inbred J tadpoles (j1 to j10) with a coverage 3 or 5 cloned PCR product sequenced per tadpole (38 total number of sequences). The sequences were aligned using BioEdit Sequence Alignment software and compared to the wild type (WT) sequence of the CRISPR/Cas9 targeted region (underlined). Deletions are indicated by a (−) and insertions and nucleotide substitution as are shown in lower case letters. Note: all sequences obtained from tadpoles j2 (5 out of 5) and j9 (3 out of 3) were identical to WT. (B) Pie chart showing the relative contributions of the different types of mutations observed among the 38 sequences. (C) Assessment of XNC10.1 gene expression by RT-PCR (38 cycles with primers specific for the α1 and α2 domain) on total RNA from the 10 tadpoles (j1 to j10) used in A. RT-PCR was also performed for XNC6.1 gene, not targeted by the CRISPR/Cas9 system, and for the house keeping gene GAPDH. As positive control (+) RNA from an age-matched untreated tadpole was used. (D) Detection of invariant Vα6-Jα1.43 transcripts by qRT-PCR in an age-matched untreated control tadpole (WT) and the 8 different CRISPR/Cas9 treated tadpoles.

As further evidence of XNC10.1 gene disruption by the CRISPR/Cas9 system, we assessed XNC10.1 gene expression by RT-PCR using primers spanning α1 and α2 domains. For 8 out of 10 examined tadpoles, no XNC10.1 transcripts were detected (Fig.2C). However, a weak expression of XNC10.1 mRNA was detectable in j6 and j7 tadpoles (Fig.2A). It is possible that this kind of mutation was insufficient to disrupt XNC10.1 gene expression or that a low fraction XNC10.1 genes were not mutated due to somatic mosaicism. The absence of XNC10.1 gene expression in 2 of 10 tadpoles, j2 and j9, is puzzling as no mutations were found in the targeted region from the recovered PCR products. It is possible that j2 and j9 had a reduced penetrance of mutation that could be detected by increasing the sequence coverage. Nevertheless, transcripts of the invariant Vα6-Jα1.43 chain of T cell receptor, which is a signature of XNC10-restricted iT cells, were undetectable in all 8 analyzed tadpoles (Fig.2D; (Edholm et al., 2013)).

CRISPR/Cas9-mediated targeting of XNC10.1 α2 domain does not affect other XNC genes

We determined the feasibility and efficiency of XNC10.1 loss-of-function mediated by the CRISPR/Cas9 system in F0 X. laevis tadpoles. However, the specificity of targeting only XNC10.1 and not other XNC genes remained to be determined. Previous studies have suggested that the Cas9 protein can accommodate some mismatches between the sgRNA and the targeted region and still induce DNA double strand breaks (Blitz et al., 2013; Cradick et al., 2013; Fu et al., 2013). Furthermore, it has been reported that the Cas9 protein is more permissive for mismatches at the 5’ end of the sgRNA rather than on the 3’end (Anderson et al., 2015). To assess potential off-target effects of the CRISPR/Cas9 system the sgRNA targeting the XNC10.1 α2 domain was designed so that if the CRISPR/Cas9 system permits mismatches, other XNC genes would be targeted as well, albeit perhaps less efficiently. Thus, we aligned the XNC α2 domains and ranked the number of total as well as 3’ end located mismatches within the site targeted by the XNC10.1 -specific sgRNA (Fig. 3A). From this ranked list, two XNC α2 domains with the least number of total mismatches (XNC6.2, XNC8.2) and two XNC α2 domains with the highest number of mismatches (XNC6.1, XNC11) were chosen for analysis. The corresponding genomic regions of the 8 analyzed tadpoles carrying XNC10.1 mutations were amplified by PCR, cloned and sequenced for the off-target effect analysis, with a 3 to 5 times coverage (Fig. 3B). Notably, alignment of all these sequences to their respective WT sequences did not reveal any mutations, demonstrating that the CRISPR/Cas9 system set to target XNC10.1 did not mutate other XNC genes.

Figure 3. Assessment of off-target effects by the CRISPR/Cas9 system targeting the XNC10.1 α2 domain.

Figure 3

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(A) Multiple alignments of XNC α2 domain sequences ranked from the lowest to the highest sequence mismatches at the 3’ end of the CRISPR/Cas9 targeted sequence. (B) Absence of mutation in the α2 regions of XNC6.2 and XNC8.2 (lowest mismatch with the 3’end targeted by the designed CRISPR/Cas9 system), XNC6.1 and XNC11 (highest mismatch with the 3’end). The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequences of a region targeted by the CRISPR/Cas9 (underlined). The numbers of sequences identical to WT per total sequence analyzed are indicated in parentheses.

Generation of XNC1 knockouts by the CRISPR/Cas9 system

Having established the convenience, efficiency, and specificity of the CRISPR/Cas9 system to study the function of the XNC10.1 gene, we decided to apply this system to a different XNC gene. XNC1 was found to be a good candidate given its relative high expression by immature thymocytes and X. laevis thymic lymphoid tumor cell lines (Edholm et al., 2014; Flajnik et al., 1993; Goyos et al., 2011). We anticipated XNC1 to be critical for the development and function of a distinct iT cell population. The Cas9 protein and sgRNA targeting the XNC1 α2 domain (Fig.1B) were injected into 1-cell embryos, following the same methodology as for the XNC10.1 α2 domain. Disrupting XNC10.1 gene expression did not significantly affected the viability of the embryos. In contrast, mutating XNC1 resulted in a marked lethality from 20 to 36 days post-fertilization, which corresponded to NF stage 47 (Fig. 4A). Sequence analysis of XNC1 targeted portion from several succumbed tadpoles confirmed the occurrence of mutations including deletions and insertions (Fig.4B). Although a more detailed study will be needed to establish a potential immune function of XNC1, these results suggest a role during larval development.

Figure 4. Generation of the XNC1 knockouts.

Figure 4

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(A) Survival of outbred (ob) developing embryos that were injected with an sgRNA targeting XNC1 and Cas9 protein. Survival and developmental stages were monitored for 50 days. Control (diamond); XNC1 knockout (black square). Statistical significance between controls and knockouts was determined using a Log-rank (Mantel-Cox) test; p<0.001. The fraction of surviving tadpoles at the end of the experiment is indicated in the parentheses. (B) Multiple alignments of the portion of the XNC1 α1 domain targeted by the Cas9 protein and sgRNA from 4 different tadpoles (ob 1, 4, 5 and 6) with a coverage of 4 to 19 cloned PCR product sequenced per tadpole. The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequence of the region targeted by the CRISPR/Cas9 (underlined). Deletions are indicated by a (−) and insertions are shown in lower case letters XNC1 α1 domain was mutated with the majority of deletions.

Generation of XNC10.1 and XNC11 double knockouts in X. laevis

Given the number of XNC genes in X. laevis, the possibility to disrupt multiple genes simultaneously would be advantageous in initial screening as some of XNC genes may have redundant functions. To explore this possibility, we targeted another XNC gene XNC11 at the same time as XNC10.1. We chose XNC11 because it also highly expressed by immature thymocytes and X. laevis thymic lymphoid tumor cell lines (Edholm et al., 2014; Goyos and Robert, 2009). Whether XNC11 plays a redundant function of XNC10.1 or is required for the development of some distinct subpopulation of iT cells remains to be determined. Accordingly, we co-injected the Cas9 protein with the sgRNA targeting the XNC10.1 α2 domain and sgRNA targeting the XNC11 α1 domain. The treatment resulted in a small but significant increase in mortality (15–20%) during development compared to dejellied control embryos (Fig. 5A). Similar effects on viability caused by Cas9 protein and sgRNA has been reported in X. tropicalis (Guo et al., 2014). Nevertheless, mutations were detected both in the XNC10.1 α2 domain and the XNC11 α1 domain and as before most of these mutations were mainly out-of-frame deletions (Fig. 5B). Of note, the efficiency of mutagenesis in the XNC10.1 α2 domain was higher than in the XNC11 α1 domain. Since lower level of mutagenesis (30-25%) was also obtained by injecting Cas9 protein with only the sgRNA targeting the XNC11 α1 domain, this is likely due to the designed sgRNA. In future experiments, we plan to design and test different sgRNA targeting the XNC11 gene and determine the most effective target region. Nevertheless, these data strongly suggest that the CRISPR/Cas9 system could be used to accelerate the functional screening of the different XNC genes as well as other large closely related gene families.

Figure 5. Generation of the double knockout of XNC10.1 and XNC11 by the CRISPR/Cas9 system.

Figure 5

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(A) The survival of XNC10.1 and XNC11 double knockouts was monitored for 30 days: Dejellied control (black diamonds); XNC10 batch 1 (black squares); XNC10 batch 2 (black triangle); and XNC10 batch 3 (black circles). Survival was statistically different between the control and each XNC10 batch (p <0.003) but not among the 3 batches as determined by a Log-rank (Mantel-Cox) test. (B) Nine outbred (ob) tadpoles treated with the Cas9 protein and sgRNA against the α2 domain XNC10.1 and the α1 domain of XNC11 were sequenced, with a coverage 3 to 9 (XNC10.1) and 1 to 7 (XNC11) cloned PCR product sequenced per tadpole. The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequence of the region targeted by the CRISPR/Cas9 (underlined). Both the XNC10.1 α2 and XNC11 α1 domains were mutated with the majority of detected mutations being deletions. Mosaicism was detected within each tadpole.

Discussion

This study provides strong evidence of the applicability of the CRISPR-Cas9 system to comparative immunology by reverse genetics in X. laevis. The efficiency and specificity of this methodology should permit the use of F0 animals for screening distinct phenotypes such as the interruption of iT cell development as well as other biological effects.

Previously, we generated transgenic X. laevis using RNAi technology. However, due to variations in the levels of RNAi-mediated silencing the residual gene expression in F0 and in further progenies may interfere with the interpretations of XNC gene functions. As in the case of other model organisms, the CRISPR/Cas9 system proved to be a feasible and convenient technology for X. laevis (Harrison 2014). Based on recent reports in X. tropicalis genome engineering, we decided to use a two-component CRISPR/Cas9 system, namely in vitro transcribed sgRNAs and the recombinant Cas9 protein (Bhattacharya et al. 2015). Instead of plasmid or mRNA-based expression, we injected the Cas9 protein that has been reported to cause less toxicity and fewer off-target effects, and higher mutation frequencies (Bhattacharya 2015). Although, the CRISPR-Cas9 system results in somatic mosaicism, the efficiency of gene editing was sufficient to obtain biological effects in F0 animals, which strengthen X. laevis tadpoles for immunological studies. This should allow us to accelerate the screen of the numerous other XNC genes described to date in F0 animals. F1 progenies will be generated once interesting genes are identified.

The potential off-target effects of the CRISPR/Cas9 system are mainly evaluated by computer software (e.g., CRISPRdirect). Since the majority of the XNC genes are clustered in a defined genomic region and display high sequence similarity, this gene family provided an ideal system to experimentally test potential off-target effects. Notably, we found no evidence of off-target effects in other XNC genes in the case of XNC10.1 knockouts. This result is of relevance since it suggests that it will be feasible to target specifically different XNC genes.

Although targeting XNC10.1 with the CRISPR/Cas9 system resulted in high frequency of mutations (80%), a significant fraction of these mutations were mosaic, ranging from 2 to 4 different mutation types per individual (Fig.2A). This somatic mosaicism cannot be accounted by X. laevis genome allotetraploidy because both the MHC and XNC loci have been diploidized (Du Pasquier et al., 1977). This is further confirmed by the analysis of the X. laevis sequence assembly (Fig. 1A). Thus, it is more likely that some of the CRISPR/Cas9-mediated genome modifications do not occur at one-cell embryo but at later developmental stages, which result in independent double strand breaks with different mutations upon imprecise DNA repair mechanism. Interestingly, similar somatic mosaicism mediated by the CRISPR/Cas9 system was recently observed in tyrosinase gene mutants, phenotypically in X. tropicalis and by deep sequencing in mouse (Blitz et al., 2013; Yen et al., 2014).

So far we have used a two-component CRISPR/Cas9 system in which the repair of broken DNA strands is conducted by an error-prone non-homologous end joining (NHEJ) mechanism. In this process, a deletion or insertions is introduced as broken DNA strands are directly ligated. In future experiments in order to avoid genetic mosaicism, we plan to utilize a three-component CRISPR/Cas9 system in which an exogenous repair template, often carried on a plasmid, induces DNA repair break by a precise homology repair (HR). Importantly, in this template along homologous sequences to the targeted gene, we will encode a small 7 bp deletion, which will be efficient enough to disrupt the gene expression. The three-component CRISPR/Cas9 system has been reported to increase the frequency of exact mutations (Gratz 2014).

In the case of XNC11 gene, we observed a lower frequency of mutagenesis. Recent reports have also noted that different sgRNAs can have various gene editing efficiencies, which could be due to a chromatin-induced position effect (Hwang 2013 and Guo 2013). One approach to alleviate this obstacle would be to design sgRNA targeting other regions of XNC11 gene.

Functional redundancy is frequent in related genes that are essential for immune responses. Here, the ease of multiplexing the mutation strategy may uncover whether a role of one XNC gene can be masked by the expression of another. We were able to generate double knockouts for both XNC10.1 and XNC11 genes, though with variable efficiencies.

In conclusion, despite high somatic mosaicism and unequal frequency of mutagenesis, we find the CRISPR/Cas9 system amenable to investigate the roles of XNC genes in X. laevis biology and immune functions.

Supplementary Material

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Highlights.

  • Conserved cluster of a large nonclassical MHC gene family in X. tropicalis & laevis

  • Retention of most clustered nonclassical MHC genes on X. laevis large chromosome 8

  • Application of the CRISPR/Cas9 system to immune gene loss-of-function in Xenopus

  • Highly specific and efficient gene disruption of Xenopus nonclassical MHC genes

  • Single and double gene knockout X. laevis were generated to facilitate future studies

Acknowledgments

We would like to thank Francisco De Jesús Andino and Tina Martin for the animal husbandry. We also thank the Cold Spring Harbor Laboratory Xenopus 2015 course and especially Drs. Karen Liu, Mustafa Khokha, and Emily Miss for excellent training and reagents. This research was supported by R24-AI-059830 grant from the National Institute of Allergy and Infectious Diseases (NIH/NIAID). M.B. was supported by a predoctoral fellowship Ruth L. Kirschstein Predoctoral F31 (F31CA192664) from the National Cancer Institute (NIH/NCI) and NIH-T32 training grant AI007285. E-S.E. was supported by the Nation Science Foundation IOS-1456213.

Footnotes

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Disclosures/Conflicts of Interest

The authors disclose no conflicts of interest.

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