RNA-Responsive gRNAs for Controlling CRISPR Activity: Current Advances, Future Directions, and Potential Applications

Abstract

CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the cell type, disease state, and environmental challenges cells are facing. Single-guide RNA (sgRNA) engineering played a major role in the development of RNA-responsive CRISPR systems. Following further optimizations, RNA-responsive sgRNAs could enable the development of novel therapeutic and research applications. This review introduces engineering strategies that could be employed to modify Streptococcus pyogenes sgRNAs with a focus on recent advances made toward the development of RNA-responsive sgRNAs. Future directions and potential applications of these technologies are also discussed.

Introduction

Recent years have seen CRISPR-Cas9 technology become one of the most essential tools for genome engineering.¹ Functional components of Streptococcus pyogenes CRISPR systems are the SpCas9, the CRISPR RNA (crRNA), and the trans-activating crRNA (tracrRNA).² crRNA plays an essential role in programming Cas9 to recognize the desired DNA sequence, while the tracrRNA is involved in mediating interactions with Cas9 (Fig. 1A).

FIG. 1.

Engineering sgRNA expression cassettes for RNA sensing. (A) crRNA base pairs with the tracrRNA, forming a functional gRNA complex. The spacer sequence within the crRNA is represented in orange, while the crRNA component complementary with the tracrRNA is represented in light gray. Spacer sequence plays an essential role in programming SpCas9 to recognize the desired DNA sequence, while the tracrRNA (dark gray) is involved in mediating interactions with SpCas9.³ (B) sgRNAs are generated by fusing crRNA and tracrRNAs, while employing a minimal tetraloop structure (cyan).³ The sgRNA sequence is composed of six functional modules: spacer, lower stem, upper stem, and nexus, as well as two hairpins. The spacer sequence has its origin in the crRNA, as well as the lower and upper stem sequences represented in light gray. Lower and upper stem sequences represented in dark gray, as well as nexus and hairpins have their roots in the tracrRNA.³⁰ (C) MREs enable sgRNA processing from polymerase II transcripts. Complementary miRNAs cleave off the 5′ cap and 3′ polyadenylation signals, resulting in the processing of functional sgRNAs.¹³ (D) Anti-CRISPR proteins are employed for miRNA sensing. In tissues expressing miRNAs of interest, anti-CRISPR genes are silenced, thus derepressing CRISPR activity.^19,20 (E) Bacterial mRNA targets could be detected using RENDR technology. RENDR transcript 1 encodes for an RBS, a split ribozyme, as well as a guide RNA sequence complementary with target mRNA. RENDR transcript 2 encodes for a guide sequence, a split ribozyme, as well as the dCas9 coding sequence. Upon recognition of the target mRNA, the two transcripts are located in close spatial proximity. This leads to the formation of a functional ribozyme and a trans-splicing reaction, which results in the fusion between RBS and the dCas9 coding sequence. Addition of RBS enables translation of dCas9 mRNA.²⁹ (F) Endogenous loci expressing lncRNAs are engineered for monitoring lncRNA transcription. This is achieved by fusing lncRNAs with tRNA scaffolds and sgRNA expression cassettes. Endogenous RNaseP and RNaseZ process sgRNA from the lncRNA sequence by recognizing tRNA scaffolds.¹⁴ crRNA, CRISPR RNA; dCas9, dead Cas9; lncRNAs, long noncoding RNAs; MREs, miRNAs responsive elements; mRNA, messenger RNA; RBS, ribosome-binding site; RENDR, Ribozyme-ENabled Detection of RNA; sgRNAs, single-guide RNAs; tracrRNA, trans-activating CRISPR RNA.

The number of CRISPR components could be further reduced by using single-guide RNAs (sgRNAs) obtained by fusing the crRNA and the tracrRNA (Fig. 1B).³ sgRNA targets Cas9 to a desired genomic location, where it creates a double-strand break (DSB). Depending on the DSB repair mechanism employed, gene knockout or precise gene editing can be achieved.¹

As CRISPR-Cas9 might cause off-target mutations,⁴ different methods were developed for precisely controlling CRISPR activity.^5–12 Proof-of-concept studies have shown that CRISPR systems could be controlled by light⁶ or inducer molecules.^7–11 Nevertheless, all these approaches require exogenous inputs. Linking CRISPR activity with the detection of endogenous biomarkers naturally expressed by target cells would open more possibilities in terms of therapeutic and research applications.

Recent studies have demonstrated that CRISPR activity could be controlled by sensing endogenous RNA molecules.^13–24 This was achieved by either engineering expression cassettes encoding CRISPR components or by designing RNA-responsive sgRNAs. An example of an engineered expression cassette involves miRNA sensing by introducing miRNA-responsive elements (MREs) within Cas9, sgRNA, or anti-CRISPR transcripts.^13,17–20 Engineered CRISPR transcripts are compatible only with DNA or RNA delivery methods. Nevertheless, delivery of Cas9-sgRNA ribonucleoprotein (RNP) complexes is often a preferred route for therapeutic applications, as the use of RNPs, which are relatively rapidly degraded, reduces off-target effects associated with prolonged Cas9 expression.²⁵

Unlike engineered expression cassettes, RNA-responsive sgRNA sensors should, in theory, also be compatible with RNP delivery. These engineered sgRNAs fold into complex secondary structures that can change the conformation upon hybridization with endogenous RNAs, resulting in either activation or inactivation of sgRNAs.

This review will introduce recent advances made toward the development of CRISPR-based RNA sensors. While we present RNA-sensing strategies through engineered expression cassettes, the review is mainly focused on RNA-responsive sgRNAs. As most published RNA sensors are based on engineered SpCas9 sgRNAs, we introduce guide RNA engineering opportunities, together with engineering strategies for designing conditional sgRNAs. In addition to published examples of RNA-responsive sgRNA designs, we discuss future directions and potential applications for these technologies.

RNA Sensing Through Engineered Expression Cassettes

When delivered as plasmids, sgRNAs are typically expressed from polymerase III promoters, but with specific modifications in the construct design, expression using polymerase II promoters is also possible.²⁶ When transcribed by polymerase II, sgRNAs are capped and polyadenylated. Although the cap structure sterically blocks Cas9 activity, endogenous factors such as Drosha can be deployed to remove the cap. For example, sgRNAs can be expressed as part of polycistronic transcripts that also encode short-hairpin RNAs (shRNAs). Subsequently, endogenous Drosha processing of shRNAs results in the release of sgRNAs and the removal of the cap.²⁷ Alternative ways of releasing sgRNAs from polymerase II transcripts include RNA interference (RNAi), self-cleaving ribozymes, site-specific nucleases, or tRNA scaffolds.²⁶ As tRNA scaffolds could also act as polymerase III promoters, minimal tRNA scaffolds without transcriptional activity can also be used.²⁸

The sensing system using engineered polymerase II cassettes was previously employed to detect endogenous miRNAs in mammalian cells. This was achieved by flanking sgRNAs by MREs (Fig. 1C). In cells that express miRNAs of interest, polymerase II transcripts are processed, resulting in the formation of active Cas9-sgRNA complexes.¹³ Moreover, the stability of Cas9-encoding transcripts was also regulated by MREs. In this way, the presence of target miRNAs could silence the expression of Cas9.¹⁷

Alternative ways of controlling CRISPR-Cas9 activity involves deploying or controlling the expression of anti-CRISPR proteins. Anti-CRISPR proteins inhibit the activity of Cas9-sgRNA complexes through different mechanisms, including stoichiometric and enzymatic inhibition and covalent Cas9 modifications or sgRNA cleavage.¹⁸ To control the expression of one such protein that blocks Cas9 activity, AcrIIA4, specific MREs were included within the AcrIIA4 transcripts. As a result, AcrIIA4 is degraded in cells expressing miRNAs of interest, resulting in a specific tune-up of CRISPR activity.¹⁹ A similar approach was used for restricting CRISPR activity to tissues of interest in mice. Neisseria meningitidis Cas9 (NmeCas9) and AcrIIC3 anti-CRISPR protein were included in two different adeno-associated viruses (AAVs). AcrIIC3 messenger RNA (mRNA) contained MREs for a liver-specific miRNA, miR-122 (Fig. 1D). After systemic co-delivery of AAV vectors, heart and liver tissues were collected, and editing was shown to occur in the liver preferentially.²⁰

mRNA sensing was also demonstrated in Escherichia coli by the Ribozyme-ENabled Detection of RNA (RENDR) technology. A dCas9 (dead Cas9 lacking endonuclease activity) expression cassette was split into two transcripts. First transcript contained a ribosome-binding site (RBS), while the second transcript consisted of the dCas9 coding DNA sequence (CDS). Split ribozyme sequences and guide sequences complementary with the mRNA to be sensed were also included within the two mRNA sequences. The two engineered transcripts bind to their target mRNA in closed proximity, leading to the formation of a functional ribozyme. A trans-splicing reaction enabled formation of a functional dCas9 expression cassette, consisting of both RBS and CDS (Fig. 1E).²⁹

Genome engineering was also employed for sensing long noncoding RNA (lncRNA). To this end, sgRNAs flanked by tRNA scaffolds were inserted within the 3′UTRs of the lncRNAs of interest (Fig. 1F). sgRNAs were transcribed together with lncRNAs and processed by endogenous RNase P and RNase Z before interacting with Cas9.¹⁴

sgRNA Engineering Strategies

The sgRNA sequence is composed of six functional modules: spacer, lower stem, upper stem, nexus, as well as two hairpins. The spacer sequence has its origin in the crRNA, while the nexus and hairpins have their roots in the tracrRNA. The lower and upper stems are formed by base-pairing interactions between the crRNA and tracrRNA molecules fused using a minimal “GAAA” tetraloop.³⁰ The position of these modules with regard to Cas9 was determined in structural studies.^31–33

A crystal structure of the Cas9-sgRNA-DNA complex revealed that the tetraloop, the loop of the second hairpin, as well as the 5′ and 3′ sgRNA ends, point outside of the Cas9 structure.³¹ Both structural^31–33 and functional experiments^3,30,34 suggested that engineering the 5′ sgRNA end, tetraloop, loop 2, and the 3′ terminus would be possible without massively disrupting sgRNA function. While Nowak et al³⁵ provided a comprehensive summary of the sgRNA engineering efforts before 2016, we will also introduce more novel approaches.

Spacer engineering was attempted by truncating (Fig. 2A) or extending (Fig. 2B) the original 20 nt sequence. Removing nucleotides from the 5′ end or replacing ribonucleotides with deoxyribonucleotides at this end reduces Cas9 off-target effects.^34,36 17–19 nt spacers were not associated with a significant reduction in cutting activity compared to original 20 nt spacers.³⁴ Reducing the spacer length below 14–15 nt inhibited Cas9 nuclease activity, but did not prevent binding to its DNA target. Truncated spacers efficiently triggered transcriptional activation.³⁷

FIG. 2.

Engineering strategies that do not compromise sgRNA activity. In this figure, sgRNA spacer sequences are represented in orange, the “GAAA” tetraloop in cyan, and the sgRNA scaffold sequences in gray. (A) Cas9-sgRNA complexes containing truncated spacers sequences (down to 17 nt) maintain their ability to cut DNA,³⁴ while Cas9-sgRNA complexes with shorter spacer truncations (down to 8 nt) still maintain their ability to bind target DNA.^37,38 (B) Extended spacer sequences that do not form complex secondary structures are truncated back to canonical 20 nt spacer sequences. Truncation is performed by an uncharacterized mechanism, likely involving cellular RNases.^41,42 (C) Examples of sgRNA scaffold engineering involve fusions with aptamer domains (dark purple)^37,46–49 or of sequences complementary with RNA sequencing capture probes (green).^52,53 (D) Examples of sgRNA 3′ end engineering involve fusion of sgRNAs with aptamer sequences (dark purple),⁴⁶ prime-editing reverse transcription (light brown), and primer-binding sites (dark brown),⁵⁷ as well as fusion of sgRNAs with target sequences complementary with RNA sequencing capture probes (green).⁵²

The average dwell time (duration of binding to DNA targets) of dCas9 is affected by the length of the spacer sequence. In mammalian cells, 11 nt spacers resulted in an average dCas9 dwell time of 206 min, whereas 8 nt spacers resulted in only 25 min of average binding time.³⁸ Mismatches within the last 5 nt of the spacer³ or substitution of RNA nucleotides with DNA nucleotides³⁶ can also be tolerated. Most of the nucleotides in the entire sgRNA sequence could be replaced with DNA nucleotides.^39,40

In a study attempting to improve Cas9 on-target specificity, 30 nt spacers tested were trimmed to 20 nt by an uncharacterized cellular mechanism.⁴¹ A similar phenomenon was observed for self-targeting sgRNAs with spacer sequences of up to 70 nt.⁴² Processing of 20 nt spacer sequences was also observed when co-expressing a crRNA array with a tracrRNA in mammalian cells. Although without definitive evidence, crRNAs are believed to be processed by type III RNases in mammalian cells,⁴³ reminiscing the crRNA maturation processes observed in S. pyogenes.² Conversely, extended spacer sequences that form loops appear more stable, as these secondary structures may protect sgRNAs from being recognized by endogenous nucleases. Extensions in which spacers are embedded within hairpins are also thought to be stable.^9,11,44,45 This includes hairpins that only partially cover the spacer sequences explored to reduce Cas9 off-target effects.^44,45

Scaffold modifications (Fig. 2C) were employed for recruiting engineered proteins to exposed sgRNA domains. MS2, PP7, and boxB are well-characterized aptamers bound by the MCP, PCP, and N22 proteins, respectively. Such aptamers were embedded into the sgRNA tetraloop and loop 2.^37,46–49 By fusing aptamer-binding proteins with transcription factors^10,21 or fluorescent proteins,^47,49,50 modulation of gene expression and imaging of repetitive DNA loci were achieved. For instance, sgRNA scaffolds were modified to improve sgRNA folding or expression yields from polymerase III promoters.⁵¹

sgRNA loops and the 3′ scaffold ends were also engineered to include A/G-rich structures that could be captured by oligo dT RNA-sequencing capture probes,⁵² as well as other target-specific probes.⁵³ Scaffold modifications also enabled designing of sgRNAs capable of targeting their own DNA expression cassette. This was achieved by replacing the first three “GUU” scaffold nucleotides with a “GGG” palindromic associated motif site. These designs enabled continuous barcode evolution for deep lineage tracing applications.^42,54

3′ end sgRNA modifications (Fig. 2D) could also recruit engineered MS2, PP7, and Com proteins to Cas9-binding sites.⁴⁶ Additional modifications include appending Cas12a guide RNAs to the 3′ end of the Cas9 sgRNA to improve co-expression of the two gRNAs at a single-cell level. These chimeric gRNAs are recognized by Cas12a that processes its own guide RNA, while releasing a functional Cas9 sgRNA.⁵⁵ CRISPR-display technology suggests that fusing the 3′ sgRNA end with lncRNA sequences is also possible.⁵⁶

Furthermore, the prime editing technology also relies on modifying the 3′ end of the sgRNA. In the prime-editing gRNAs (pegRNA), a template for reverse transcription and the binding site for reverse transcriptase are both fused to the 3′ end of the scaffold.⁵⁷ Similar to extended spacer sequences, unprotected 3′ end sgRNA extensions also appear to be degraded. In a subsequent optimization of pegRNA designs, structured RNA motifs were appended to the 3′ end of the engineered pegRNAs for promoting their stability.⁵⁸

Engineering Strategies for Designing Conditional sgRNAs

In contrast to nondisruptive sgRNA engineering, conditional sgRNA designs employ engineering strategies that interrupt sgRNA activity. A study was carried out to identify secondary structure domains that are essential for sgRNA activity (Fig. 3A). Blocking the functional sgRNA domains using single-stranded DNA probes demonstrated that interfering with spacer and lower stem, nexus, as well as hairpin 1 considerably inhibits sgRNA activity, while blocking hairpin 2 does not cause a substantial inhibition.¹²

FIG. 3.

Conditional sgRNA designs. In this figure, sgRNA spacer sequences are represented in orange, the “GAAA” tetraloop in cyan, and the sgRNA scaffold sequences in gray. (A) sgRNA function could be abolished by short nucleic acid probes complementary with important functional domains (green). Probes complementary with spacer and lower stem, nexus, and hairpin 1 abolish sgRNA activity. Probes complementary with hairpin 2 do not substantially abolish sgRNA activity.¹² (B) Light-inducible crRNAs incorporate photocleavable groups (purple) within the spacer sequence. In the absence of light, these groups prevent gRNA complexes from recognizing their DNA targets. Exposure to light results in the removal of caging groups and gRNA activation.⁶ (C) Theophylline-inducible sgRNAs form a complex secondary structure in the absence of theophylline. In this design, the sgRNA spacer sequence is fused with a theophylline-inducible ribozyme (blue) as well as a sequence complementary with the spacer sequence (green). Theophylline (red) binding triggers a structural configuration resulting in ribozyme-mediated removal of the spacer-blocking sequences.¹¹ (D) Csy4-inducible sgRNAs contain a Csy4 recognition sequence (dark blue) as well as a sequence that base pairs with the spacer sequence (green). Csy4 nuclease (purple) cleaves its recognition sequence, resulting in sgRNA activation.⁹ (E) p53-inducible sgRNA becomes activated following a conformational change induced by p53 (red) binding to its aptamer (yellow). This configuration change displaces the spacer-blocking sequence (green), leading to sgRNA activation.⁷ (F) ASO-inducible sgRNA is activated by single-stranded DNA ASO (dark brown). sgRNA designs contain a loop sequence (light orange) as well as a sequence that blocks the spacer (green). ASOs base pair with the sgRNA loop structure, leading to the formation of DNA-RNA hybrids. RNase H (light brown) recognizes RNA-DNA hybrids, leading to sgRNA processing and formation of canonical 20 nt spacers.⁹ ASO, antisense oligos.

As CRISPR-Cas9 might cause off-target mutations,⁴ different methods were developed to precisely control sgRNA activity.^6–12 Chemical sgRNA modifications enabled conditional sgRNA activation following exposure to light (Fig. 3B).⁶ Alternative sgRNA activation triggers included small molecules such as theophylline (Fig. 3C),^7,8,10,11 tetracycline,⁷ or guanine.¹¹ Small molecule sensing was achieved by embedding specific aptamers within the sgRNA structure. Site-specific endoribonucleases such as Csy4 were also shown to activate modified sgRNAs in mammalian cells (Fig. 3D).⁹ However, all these approaches for conditional sgRNA engineering require additional exogenous inputs. CRISPR-based sensors capable of harvesting endogenous information to control Cas9 activity would massively benefit the development of therapeutic and research applications.

Early studies have demonstrated that sgRNA activity can be controlled by endogenous proteins upregulated in cancer cells. Examples included proteins such as p53, NF-κB, AFP, B-catenin, and HSF1, for which well characterized high-affinity aptamers are available. These sensors were generated by extending the 3′ sgRNA ends with sequences complementary to the spacer. Specific aptamers were also included within 3′ extensions (Fig. 3E). In cells without high expression of target proteins, 3′ end extensions blocked the spacer, resulting in a nonfunctional sgRNA. However, in cells overexpressing target proteins, aptamer-protein interactions created a conformational change that exposed the spacer sequence and activated the sgRNA.⁷

As high-affinity aptamers are not available for all endogenous factors, a different approach could involve controlling sgRNA activity by endogenous RNAs. Several studies suggested that single-stranded DNA (ssDNA) oligos could activate sgRNAs whose activity was inhibited by secondary structures.^9,12,59 Activation of such engineered sgRNAs called iSBH-sgRNAs (inducible spacer-blocking hairpin sgRNAs) was thought to depend on RNaseH hydrolysis of RNA molecules when hybridized to DNA (Fig. 3F).⁹ Apart from ssDNA, single-stranded RNA (ssRNA) could also activate engineered sgRNAs. Interestingly, ssRNAs have been reported to lead to better activation outcomes than ssDNA sequences in vitro. This was believed to be because double-stranded RNA (dsRNA) duplexes are more thermally stable than DNA-RNA hybrids.⁵⁹

RNA-Responsive sgRNA Designs

RNA strand displacement is a molecular mechanism in which an ssRNA invader attacks a preformed RNA duplex. A conformational change occurs as the ssRNA hybridizes to one of the RNA duplex strands, leading to the displacement of the second duplex strand.⁶⁰ Starting from this principle, several OFF-ON (Fig. 4) or ON-OFF (Fig. 5) sgRNA switches were engineered for sensing RNA triggers. In most cases, this was achieved by engineering sgRNAs to fold into complex secondary structures. Both synthetic and endogenous ssRNA triggers can act as invaders and disrupt secondary structures within engineered sgRNAs. Several proof-of-concept experiments showed that such systems could be applicable for sensing RNA in vitro.^59,61–63 Sensing synthetic triggers,^23,63–66 RNAi effectors,^{16,21,65,67,68} and mRNAs^{15,16,21,23,65} was demonstrated in live cells, including bacteria and mammalian cells.

FIG. 4.

OFF-ON sgRNA switches. RNA-responsive OFF-ON sgRNA switches are designed to fold into complex secondary structures that, in the ground state, inhibit sgRNA activity. Upon recognizing complementary RNAs, engineered sgRNAs become activated. Figures A-I represents published engineering strategies for designing OFF-ON sgRNA switches. sgRNA modifications are represented in dark blue, while complementary RNA triggers are represented in light blue. Spacer sequences are represented in orange, the “GAAA” tetraloop in cyan, and the sgRNA scaffold sequences in gray (A) 5′ Extension × spacer × 5′ side toehold design.⁵⁹ (B) Toehold switch conditional sgRNA.⁷⁰ (C) Toehold-gated sgRNA.²¹ (D) RNA-interacting sgRNA.²³ (E) Survivin-sensing sgRNA.¹⁵ (F) Interdomain × [3′ side stem bulge + hairpins] design.⁵⁹ (G) 3′ Extension × hairpins design.⁵⁹ (H) Activatable sgRNA.⁶² (I) mRNA-sensing sgRNA.⁶¹

FIG. 5.

ON-OFF sgRNA switches. RNA-responsive ON-OFF sgRNA switches are designed to fold into a secondary structure that is compatible with sgRNA activity and CRISPR function. Upon recognizing complementary RNA triggers, engineered sgRNAs become inactive, repressing CRISPR function. (A–C) Represent published engineering strategies for designing ON-OFF sgRNA switches. sgRNA modifications are represented in dark blue, while complementary RNA triggers are represented in light blue. Spacer sequences are represented in orange, the “GAAA” tetraloop in cyan, and the sgRNA scaffold sequences in gray (A) Repressible sgRNA.⁶² (B) Terminator switch conditional sgRNA.⁶⁴ (C) Splinted switch conditional sgRNA.⁶⁴

OFF-ON sgRNA switches are generated by engineering diverse components of the sgRNAs, to generate secondary structures incompatible with sgRNA function. This can be achieved, for instance, by interfering with the spacer sequences (Fig. 4A–E, H),^{15,21–23,59,62,64–66} or preventing the formation of essential scaffold domains (Fig. 4F, G, I).^59,62 Cognate RNA triggers delivered exogenously or sensed endogenously initiate strand displacement reactions that result in the formation of active sgRNAs.

ON-OFF sgRNA switches are functional in the absence of a cognate RNA trigger. The introduction of cognate triggers results in the formation of secondary structures that are incompatible with the sgRNA function. Such designs are based on the formation of dsRNA sequences upstream the spacer sequence (Fig. 5A)⁶² or insertion of trigger-complementary sequences within the tetraloop and loop 2 (Fig. 5B, C).⁵⁹

Sensing of RNA Triggers In Vitro

In vitro sensing of RNA triggers was demonstrated in several publications.^{24,59,61–63,69} Jakimo et al proposed a series of designs (Fig. 4A, F, G) that sensed short synthetic RNA triggers.⁵⁹ dsRNA structures formed immediately upstream of the spacer sequence inhibit Cas9 activity. From this observation, both activatable sgRNA (Fig. 4H) and repressible sgRNA (Fig. 5A) designs were generated. Activatable sgRNAs have complementarity between 5′ and 3′ sgRNA ends, while repressible sgRNAs are sgRNAs with extended spacers. When the supernumerary nucleotides in repressible sgRNAs hybridize with RNA triggers, Cas9 activity is repressed.⁶² Even though the repressible sgRNA design works in vitro, it will be essential to test it in living cells, given that previous studies have suggested that extended spacer sequences get truncated by RNases.^41,42

mRNA-sensing sgRNA (Fig. 4I) designs involve forming a secondary structure between the sgRNA tetraloop and the 3′ end of sgRNA, including an unpaired single-stranded toehold that promotes hybridization with incoming triggers and facilitates strand displacement. The mRNA-sensing sgRNA secondary structure is incompatible with the sgRNA function in an OFF state, and strand displacement activates the sgRNA. This design was tested using in vitro Cas9-cleavage assay activated by different fragments of the mCherry mRNA. Short mCherry fragments (80 nt) led to 77% cleavage activity, while full-length mCherry (711 nt) resulted in only 5% cleavage activity. This study hypothesized that the longer the mRNA, the more secondary structures it forms, thus preventing sgRNA strand displacement.⁶¹ Similar findings were reported for engineered Cas12a gRNAs. Sensing of mRNA sequences was also tested in vitro, but strand displacement kinetics was much slower for full-length mRNAs than shorter mRNA fragments.⁶³

tracrRNA modifications enable the designing of other types of OFF-ON switches. Recent studies have shown that SpCas9^24,62 and Campylobacter jejuni Cas9 (CjeCas9)⁶⁹ tracrRNAs could be extensively modified without compromising Cas9 activity. For SpCas9, tracrRNA positions affecting interactions between tracrRNA and crRNA were identified and sequences complementary to mRNA triggers were introduced within tracrRNA at these sites. When modified tracrRNAs hybridized with mRNA triggers, crRNAs were processed from the trigger sequences, resulting in the formation of functional crRNA-tracrRNA complexes (Fig. 6A).²⁴ A similar approach was also used for CjeCas9, and both systems were successfully employed for in vitro detection of SARS-CoV-2 RNA fragments.^24,69

FIG. 6.

Other OFF-ON gRNA switches. (A) tracrRNAs were modified to include sequences complementary with mRNA triggers. When modified tracrRNAs hybridize with mRNA triggers, mRNA triggers are processed into crRNAs. This results in the formation of functional crRNA-tracrRNA complexes.²⁴ (B) sgRNA was inactivated by removing terminal hairpins. Recognition of RNA triggers restored hairpin 1, leading to sgRNA activation. In this design, sgRNA activation was also facilitated by fusing RNA trigger sequence with sgRNA hairpin 2.⁷⁰ (C) Sensing miRNAs using two RNA hairpins and modified sgRNAs. Target miRNA (orange) binds to a complementary sequence within hairpin 1 (yellow). Displaced hairpin 1 strand (green) hybridizes to a complementary sequence within hairpin 2 (green), leading to displacement of the RNA trigger (light blue). RNA trigger hybridizes to the spacer-blocking region of the modified sgRNA (dark blue), resulting in sgRNA activation.²² (D) Endogenous mRNAs or small RNAs bridge connection between engineered crRNA′ and tracrRNA′ forming a functional gRNA complex.¹⁶ (E) Toehold-gated sgRNAs are expressed from polymerase II promoters to facilitate cytoplasmic export. Toehold-gated sgRNAs are processed in the cytoplasm, where miRNAs facilitate removal of the cap and polyadenylation signals. In the cytoplasm, complementary mRNA sequences promote sgRNA activation.⁶⁵

Sensing Synthetic RNA Triggers in Cellular Systems

Conditional sgRNAs with OFF-ON and ON-OFF logics were tested in E. coli and mammalian HEK293T cells.^61,64 Terminator (Fig. 5B) and splinted (Fig. 5C) designs of ON-OFF switches contain RNA-sensing loops at different positions within the sgRNA, and these loops do not impact normal Cas9 function (ON-state). The binding of a trigger RNA leads to the formation of a distorted sgRNA structure incompatible with sgRNA function, leading to an OFF-state. The terminator design has a single loop that hybridizes with RNA triggers, while the splinted design has two complementary loops. All three designs sensed short RNA triggers expressed at high levels in E. coli.

The terminator design was also able to sense short, nuclear RNA triggers expressed from U6 promoters in HEK293T cells.⁶⁴ As OFF-ON switches relying on blocked spacer sequences (Fig. 4B) still displayed an undesired activity in the OFF-state, a new OFF-ON switch was proposed by splitting the sgRNA structure.

In this design, hairpin 1 was truncated, and the hybridization of the RNA trigger enabled the formation of a functional gRNA (Fig. 6B). Further optimizations of conditional sgRNA switches were enabled by flanking the synthetic RNA triggers with protective hairpin structures.⁷⁰ Although helpful with sensing synthetic RNA triggers, such protective structures cannot be added to endogenous RNA species to facilitate their detection. mRNA-sensing sgRNAs (Fig. 4I) were also tested in HeLa cells, whereby in vitro transcribed 80 nt RNA triggers were co-transfected with engineered sgRNAs, resulting in a measurable modulation of Cas9 activity.⁶¹

Sensing Endogenous RNAs in Cellular Systems

RNA species used as endogenous sensed triggers include RNAi effectors (bacterial sRNA and mammalian miRNA) and mRNA. In E. coli, toehold-gated sgRNAs (Fig. 4C) detected plasmid-encoded or endogenous small RNAs (sRNAs) such as micF, ryhB, and oxyS.²¹ Lin et al proposed another mechanism for detecting sRNAs or mammalian miRNAs that uses RNA circuits containing two hairpins and a modified sgRNA. sgRNA is engineered to adopt an OFF state by appending a 3′ extension that hybridizes with the spacer and a toehold that facilitates strand displacement. Sensing the miRNA target opens the first hairpin, leading to the opening of the second hairpin, which triggers a strand displacement reaction that activates the sgRNA (Fig. 6C). Such a tri-component complex circuit design avoids sequence dependencies between the sgRNA and RNA trigger. This system enabled micF and ryhB sRNAs sensing in E. coli and mir-17, mir-16, and let-7 miRNAs in HEK293T cells.²²

mCherry mRNA was also sensed in E. coli using toehold-gated sgRNAs (Fig. 4C). Three different toehold-gated sgRNAs were designed for sensing specific subsequences of the mCherry transcript. sgRNA activation efficiencies were measured using CRISPR interference assays, and the best design resulted in up to 60% repression of a fluorescent reporter gene.²¹ RNA-interacting sgRNA designs (Fig. 4D) can also be used to sense mRNA. When sensing short transcripts in E. coli, this system yielded up to 26-fold induction/upregulation of fluorescence reporter.

However, when full mRNA sequences were used as triggers, activation dropped to only 2.5-fold for mKate fluorophore and 10-fold for the HIV infectivity factor. These numbers further confirm that the efficiency of RNA-sensing drops with the length of the mRNA being sensed.²³ RFP mRNA and genes from the arsenic operon were also sensed in bacteria using modified tracrRNAs (Fig. 6A). Engineered tracrRNAs hybridized to the mRNAs to be sensed, leading to production of crRNAs from bacterial mRNA species.²⁴

An alternative design proposed in E. coli involved truncated crRNAs (crRNA′) and tracrRNAs (tracrRNA′) that lost their ability to recognize each other through base-pairing interactions. 3′ end of the crRNA′ and 5′ end of the tracrRNA′ were fused to sequences complementary with target RNAs to be detected. Target RNAs bridged interactions between crRNA′ and tracrRNA′, resulting in the formation of a functional gRNA complex (Fig. 6D). This approach was successfully used for sensing bacterial endogenous mRNA and small RNA species.¹⁶

In mammalian cells, RNA detection is dependent on the RNA polymerase transcribing RNA triggers. Triggers expressed from polymerase II promoters were detected 3 fold less efficiently than triggers expressed using polymerase III promoters.⁶⁶ Nevertheless, several studies reported detection of polymerase II-expressed mRNA.^15,65

Expression of survivin mRNA was monitored in different cell lines, including MCF-7, HeLa, and MB-435s. In this study, the sgRNA engineering and strand displacement-mediated mRNA sensing was coupled with other engineering strategies for signal amplification. The sgRNA fold adopted in this study involved blocking the spacer sequence by a 12 nt dsRNA-blocking sequence and including a toehold gate to promote hybridization with incoming triggers (Fig. 4E). mRNA sensing was monitored by dCas9-VPR transcriptional activators and a reporter system, including eight CRISPR-binding sites upstream of a fluorescence reporter comprising five fluorescent aptamer repeats.

The survivin mRNA is highly expressed and is relatively short (429 bp). Modulators of survivin expression were used to tune mRNA copy number. Calibration curves observed suggested that 120 mRNA copies/cell was the minimum detection limit for this system.¹⁵ However, data presented in this study relied on a small number of microscopy images. And it is, therefore, not clear in which percentage of cells this technology might work and what the background activity of the reporter system is.

As mRNA is exported in the cytoplasm, another study reasoned that translocating engineered sgRNAs to the cytoplasm could improve mRNA detection. This was achieved by expressing toehold-gated sgRNAs under the control of polymerase II promoters and by using miRNA recognition elements to release these sgRNAs into the cytoplasm (Fig. 6E). Activation of such designs requires presence of miRNA targets as well as presence of the mCherry mRNA. The presence of both RNA targets caused a 2.1-fold change in CRISPRa activity, as measured using fluorescence reporters.⁶⁵ Nevertheless, certain applications might require higher dynamic ranges for endogenous RNA detection. Therefore, extra work should be done for improving activation in the presence of endogenous transcripts in eukaryotic cells.

Future Directions Toward RNA Sensing in Eukaryotic Cells

In recent years, significant progress has been made toward RNA sensing in vitro or prokaryotic systems using different RNA-sensing sgRNA designs. Some of these designs were already adopted to develop diagnostics.^24,69 The development of RNA detection platforms based on Cas9 and engineered sgRNAs could enable sensing of disease-associated RNAs, including viral RNAs. Implementation of RNA logic gates is also possible using strand displacement -based CRISPR systems.^61,62,64 Logic gates could increase the confidence of diagnostics by simultaneously analyzing two or more relevant RNA biomarkers. Furthermore, methods for amplifying signals in Cas9 in vitro cleavage assays were also proposed.²⁴ Even though existing Cas13 or Cas12 diagnostics^71–75 are promising, having alternative platforms that rely on different components would be beneficial in a pandemic. Developing diagnostic tests that differ significantly would avoid supply chain bottlenecks.

Engineered sgRNAs can also sense endogenous RNAs in bacteria. Bacteria are optimal for developing RNA-inducible sgRNAs, as they lack nuclear compartmentalization and co-transcriptional mRNA processing. Proof-of-concept bacterial applications involved the development of a whole-cell arsenic biosensor.²⁴ Alternative applications in bacteria could involve synthetic gene circuits for metabolic engineering or diagnostics. Furthermore, we envision that developing reliable sgRNA designs that could sense endogenous RNAs in eukaryotic cells would open many possibilities for novel therapeutic and research applications.

Proof-of-concept studies suggest that engineered sgRNAs can sense miRNA^22,65 or mRNA^15,65 in eukaryotic cells. However, further work is required for optimizing such technologies. Research is needed to understand the range of endogenous RNAs that could be sensed, determine endogenous RNA subsequences that would make good triggers, establish design rules, and understand potential off-target effects.

As a first step, it is imperative to understand the range of endogenous RNAs that could be sensed in eukaryotic cells with high efficiencies. Eukaryotic cells express many classes of transcripts differing in size, expression level, and subcellular localization. Sensing mRNA would pose a different set of challenges compared to sensing lncRNAs, miRNAs, or transcripts synthesized by RNA polymerase III.

Several kinetic parameters and mathematical models of sgRNA interactions with Cas9 and template DNA search are available.^39,76–82 Such models could be adapted to simulate the behavior of CRISPR-based RNA sensors. This would enable users to assess the feasibility of sensing a given transcript using different sgRNA designs and different concentrations of Cas9 and engineered sgRNA. Furthermore, interactions between engineered sgRNAs and Cas9 in the presence or absence of the RNA trigger, Cas9 effectors of choice, and reporter systems may influence RNA-sensing outcomes. Ultimately, the maximum level of functional Cas9-sgRNA complexes that could be formed depends on the endogenous level of RNA triggers.

Selecting Appropriate Triggers

Determining endogenous RNA subsequences that would make good triggers is another crucial step toward engineering RNA-sensing sgRNAs. Principles that emerged from designing RNA-targeting hybridization probes could facilitate the developments in this field. The position at which hybridization occurs is essential, as mRNA secondary structures and RNA-binding proteins likely impede probe binding. Several studies sought to identify probes that hybridize with open RNA structures.^83–89 In vitro experiments have shown that 20 nt oligos hybridize with different efficiencies to different subsequences within a target mRNA.⁸⁷ Similar results were reported for Cas13 tiling experiments, where well-performing gRNAs preferentially hybridized with specific mRNA subsequences.⁹⁰

5′ and 3′ mRNA ends are good starting points for designing short RNA hybridization probes such as molecular beacons (MB) or antisense oligos (ASOs). MBs are nucleic acid structures that form a hairpin and are used for RNA visualization in fixed or live cells. These probes become fluorescent following recognition of their RNA target. In an OFF configuration, MBs form a closed structure where a detection fluorophore is inhibited by a quencher. Upon binding of its RNA target, the hairpin structure opens up and a fluorescence signal is produced due to the spatial separation between the fluorophore and quencher.⁹¹

Good MB designs hybridize with start or stop mRNA codons. Shifting MB position with 8–10 nt upstream or downstream from the start and stop codons drastically reduced efficiency of RNA detection.⁸³ Similar results were reported for translation-blocking ASOs that bind to mRNA for inhibiting protein translation. ASOs successful at blocking translation hybridized 7–8 bp upstream from the start codon, overlapping with the Kozak sequence. Best translational repression outcomes were achieved using longer ASOs that covered the cap structure, the Kozak sequence, and the AUG codon.⁸⁶

Alternative approaches for identifying structurally open RNA subsequences include using in silico prediction tools such as NuPACK⁹² or ViennaRNA.⁹³ Although not perfect, in silico predictions are a good start for choosing accessible targets. Sequences predicted by NuPACK as being inaccessible also appeared inaccessible in experimental assays. However, sequences predicted by NuPACK as accessible had variable accessibilities when tested experimentally in vitro.⁸⁷ Sequencing protocols developed for mapping RNA secondary structures^94–96 may also offer a significant benefit in choosing appropriate trigger subsequences, while considering the structure of a given RNA within a cellular context.

Development of sgRNA Design Rules

Establishing good rules for designing sgRNA would significantly benefit the field. Several sgRNA designs^{15,16,21,23,59,61–68} were proposed for RNA sensing, but these designs were tested in different conditions and against different RNA triggers. A head-to-head comparison between the performance of these designs is currently lacking, and it is hard to pinpoint a design that performs better than others. Specific designs have a clear advantage, as they enable users to sense any given RNA trigger and direct Cas9 to any DNA target of choice.^{16,22,23,61–63,65,70} This was made possible by decoupling sgRNA sensing from the spacer modules.

Nevertheless, it is unclear whether design rules proposed in the literature are generalizable for other RNA triggers and spacer sequences. As suggested by several studies,^66,70 OFF-ON switches relying on secondary structures that block functional sgRNA components may not be completely silent in an OFF state. Design rules were proposed for toehold-gated sgRNAs in mammalian cells⁶⁶; nevertheless, parameters such as the energy of sgRNA folding and GC-content may help further improving sgRNA switches. High-throughput studies combined with machine learning approaches will be required for identifying optimal design rules.

Furthermore, the length of the sgRNA-sensing region might increase the affinity of the sgRNA for its endogenous RNA trigger, thus improving sgRNA activation. Synthetic ADAR guide RNAs could recruit endogenous ADAR enzymes to mRNAs of interest, resulting in RNA editing.^97–99 Progressively longer ADAR gRNAs ranging from 40 to 151 nt were tested, and longer gRNAs were shown to cause better editing outcomes.⁹⁷ Similar results were reported for Reprogrammable ADAR Sensors (RADARS)¹⁰⁰ and trans-splicing RNAs,¹⁰¹ suggesting that longer hybridization probes might outcompete RNA secondary structures within endogenous RNAs.

Off-Target Effects

A common off-target effect of sgRNAs is directing Cas9 to nondesired genomic DNA regions.¹⁰² On top of that, RNA-sensing sgRNAs could have additional off-target effects due to nonspecific sgRNA activation. Alternative factors that could change the sgRNA configuration include other RNAs with similar sequences or poor sgRNA designs that do not fold into perfect switches. Good experimental controls should be put in place to ensure that observed Cas9 activity is linked to sensing the RNA trigger of interest.

The formation of long RNA duplexes may be a concern in eukaryotic cells. Dicer, Drosha, and AGO proteins are endogenous factors involved in the maturation of miRNAs. Such factors recognize and cleave specific dsRNA configurations.¹⁰³ Furthermore, ADARs are also involved in the modification of dsRNAs.¹⁰⁴ It would therefore be important to assess whether such factors might cleave or process dsRNAs formed by base pairing between cellular RNAs and modified sgRNAs and if these molecular events might impact the integrity of RNA triggers to be sensed.

Another potential concern regarding the formation of dsRNA structures in eukaryotic cells is activating the type I interferon (IFN) response. IFN response is an innate immune mechanism involved in defense against RNA viruses. dsRNAs are recognized by RNA sensors such as RIG-I, MDA5, and TLR3.¹⁰⁵ After dsRNA detection, a signaling cascade is triggered, leading to nonspecific degradation of cellular RNAs and translation arrest. In this way, cells attempt to block viral replication and synthesis of viral components.¹⁰⁶ IFN response was also associated with cellular toxicity following delivery of in vitro transcribed sgRNAs with 5′ triphosphate groups¹⁰⁷ or RNAi effectors in mammalian cells.¹⁰⁸

Testing whether dsRNA duplexes resulting from sgRNA interaction with endogenous RNAs trigger IFN responses would be necessary, while optimizing this technology. If type I IFN gets activated, additional engineering could prevent this. Chemical modifications such as O-Me or the addition of fluoride to the 2′ ribose position could be used for avoiding TLR activation. Adenosine modifications work best for siRNAs and reduce immune stimulation without compromising siRNA efficiency.¹⁰⁹

Potential Applications in Eukaryotic Cells

CRISPR therapeutic strategies

Following further optimization and characterization of RNA-sensing sgRNAs, this technology could open novel possibilities for CRISPR therapeutic and research applications. CRISPR therapeutic strategies might benefit from developments in RNA-sensing sgRNAs. Several therapeutic approaches have been proposed to date, involving both ex vivo and in vivo genome editing.^25,110 Nevertheless, CRISPR-Cas9 editing was shown to cause off-target effects by promoting chromosomal rearrangements and deletions.⁴

Some strategies for minimizing such effects presented in the literature involve reducing off-target effects by controlling the duration of Cas9 activity and attempting targeted delivery of CRISPR components to disease-affected tissues.^5,110 Some commonly selected strategies for achieving this involve using AAVs with preferential tissue tropism or targeted nonviral vectors. Nonviral vectors such as lipid nanoparticles, polymers, or cell-penetrating peptides typically involve the delivery of Cas9-sgRNA RNPs for reducing off-target effects associated with prolonged Cas9 expression.²⁵ Other approaches involve the use of AAVs that drive the expression of CRISPR components under the control of tissue-specific promoters.¹¹¹

For cell types or diseased cells where finding precise surface biomarkers or tissue-specific promoters could be challenging, using RNA biomarkers to activate Cas9 activity might represent an excellent alternative solution.²⁰ Novel therapeutic strategies could combine existing targeted or nontargeted delivery methods with CRISPR-based RNA sensors for increasing the specificity of such therapeutics. For example, CRISPR-based RNA sensors could be delivered to cells in an OFF state. Once inside a cell, these sensors could sense the presence of transcript biomarkers and then only activate their therapeutic function.

Depending on the CRISPR effector of choice, RNA sensing would enable conditional gene editing or modulation of gene expression. Using a standard Cas9 nuclease, conditional gene knockouts or precise genome editing would become possible following the induction of DNA DSBs.¹ Using other engineered Cas9 versions would enable expanding the potential applications of this technology. Examples include base-editing modules,^112,113 prime editors,⁵⁷ transcriptional activators (dCas9-TA),^49,114,115 or transcriptional repressors.¹¹⁶

Future research developments and applications

Coupling RNA sensing with CRISPR transcriptional activators would enable the fluorescent labeling of cells expressing transcripts of interest. Such approaches could offer an alternative to existing RNA-sensing methods. Methods such as hybridization chain reaction,^117–131 fluorescent aptamers,^{117,132–137} or fluorescent RNA-binding proteins^{95,117,138–146} enable visualization of RNA foci as well as their subcellular localization. Nevertheless, such methods do not allow the linking of RNA sensing to any downstream application. For example, fluorophores could be replaced with proteins such as ubiquitin ligases^65,66 or biotin ligases. Ubiquitin ligases conjugated with antibodies could promote target protein degradation^65,66 in cells of interest, while biotin ligases could enable labeling of RNAs or proteins^147,148 for downstream purification and analysis.

By analyzing the cellular transcripts and their levels, information could be extracted about the cell identity and the environmental challenges the particular cells face.^149,150 Potential research applications of CRISPR-based RNA sensors include restricting the activity of CRISPR to different tissues or developmental stages⁶⁴ or cells in which specific pathways are turned on. Examples could consist of cells with active DNA damage repair mechanisms, cells at different cell cycle stages, cells that upregulate specific metabolic pathways, and so on. Nuclease Cas9 has been previously used for lineage tracing experiments.¹⁵¹ CRISPR-based RNA sensors might enable the restriction of such lineage tracing experiments to cells that express RNA biomarkers of interest.

Conclusion

To sum up, several RNA-responsive gRNA designs were described in the literature. This includes both ON-OFF and OFF-ON switches. So far, the applications of RNA-responsive gRNAs only involved the development of in vitro diagnostic tools or bacterial gene circuits. Despite some proof-of-concept studies, further work is necessary for implementing these systems in eukaryotic systems. Following additional optimizations, this technology could open novel possibilities in terms of CRISPR therapeutic and research applications. Further advances in RNA-sequencing technologies and a better understanding of the RNA biomarkers that define cell types and cell states would highly benefit the development of RNA-responsive gRNAs. Choosing appropriate RNA biomarkers would be a must toward restricting CRISPR activity to cell populations of interest.

Authors' Contributions

O.P. contributed to the conception of the review and wrote the first article draft. T.A.F. and T.S.S. contributed to the conception of the review as well as structuring and editing of the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Oana Pelea was funded by the EPSRC & BBSRC Centre for Doctoral Training in Synthetic Biology, University of Oxford (Grant EP/L016494/1), EvOX Therapeutics, and Wadham College. Tatjana Sauka-Spengler is a Wellcome Trust Senior Research Fellow (215615/Z/19/Z).