CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.     

Advances in CRISPR-Cas systems for RNA targeting,tracking and editing

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.     

Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

It is important to control CRISPR/Cas9 when sufficient editing is obtained. In the current study, rational engineering of guide RNAs (gRNAs) is performed to develop small-molecule-responsive CRISPR/Cas9. For our purpose, the sequence of gRNAs are modified to introduce ligand binding sites based on the rational design of ligand–RNA pairs. Using short target sequences, we demonstrate that the engineered RNA provides an excellent scaffold for binding small molecule ligands. Although the ‘stem–loop 1’ variants of gRNA induced variable cleavage activity for different target sequences, all ‘stem–loop 3’ variants are well tolerated for CRISPR/Cas9. We further demonstrate that this specific ligand–RNA interaction can be utilized for functional control of CRISPR/Cas9 in vitro and in human cells. Moreover, chemogenetic control of gene editing in human cells transfected with all-in-one plasmids encoding Cas9 and designer gRNAs is demonstrated. The strategy may become a general approach for generating switchable RNA or DNA for controlling other biological processes.     

A simple,flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes(Sp Cas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA(sg RNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures:(1) annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and(2) ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.     

A m6A Sensing Method by Its Impact on the Stability of RNA Double Helix

N⁶‐Methyladenosine (m⁶A) is one of the most important RNA modifications in epigenetics. The development of detection method for m⁶A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m⁶A modification and target RNAs without m⁶A modification. We verified the fact that the presence of m⁶A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m⁶A detection methods in the future.     

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation,target binding and cleavage

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9''s ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9''s off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.     

Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene

The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.The CRISPR-Cas9 system is a revolutionary technology for gene targeting in cells (1–3). It consists of two components: a guide RNA and the Cas9 endonuclease that respectively pairs with the target sequence and then cleaves it (4–6). The guide RNA contains 19 nt that in theory can be custom designed to target almost any sequence in a genome (5–8). In practice, the effectiveness of the predicted guide RNAs varies dramatically (8). This problem, when compounded by other commonly encountered problems such as poor efficiency of DNA transfection or low titer of viruses, can make the isolation of successfully targeted cells a really laborious task (9). Low transfection efficiency can be improved by special techniques like nucleofection (6), but they are expensive and not readily available to all labs. A common method to enrich successfully targeted cells is to use drug resistance or green fluorescent protein (GFP) markers to select for cells that have integrated the guide RNA-expressing DNA into chromosomes (7,10). Fluorescence-activated cell sorting (FACS) sorting only enriches cells expressing high levels of GFP but not necessarily Cas9, while integration of foreign DNA, especially viral DNA, into the chromosome is itself an alteration to the genome and might cause oncogenic transformation. In some cases, Cas9 is also integrated into the genome and constitutively expressed (7). Persistent expression of the guide RNA in combination with Cas9 might lead to increasing probabilities of off-target cleavages over long-term culturing (11). In addition, if no targeted cells are obtained, it is difficult to track down the cause of failure. The guide RNA might be ineffective or the correctly targeted cells might have died off and only cells not expressing the guide RNA might have survived.To overcome these shortcomings, we have developed a transient plasmid DNA transfection-based method to enrich successfully targeted cells without the need for DNA integration into chromosomes by co-targeting the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene. This gene encodes a protein that catalyzes the conversion of hypoxanthine to inosinemonophosphate and guanine to guanosine monophosphate in the non-essential purine salvage pathway (12). HPRT⁺ cells are sensitive to 6-thioguanine (6-TG), which can be converted to the nucleotide form by HPRT and incorporated into DNA by DNA polymerase, killing cells by a process involving postreplicative mismatch repair (13–15). The strategy is to transfect cells with two plasmids that express respectively a HPRT guide RNA and a guide RNA for the gene of interest. Cas9 can be expressed from the gene on a separate plasmid, a plasmid carrying the HPRT gRNA or integrated into the chromosome (if such a cell line is already available). If a cell becomes resistant to 6-TG, it would suggest that this cell should also be competent to target the gene of interest as long as the gRNA is effective. Thus if the targeted gene is not altered in the resulting 6-TG resistant cells, it would suggest that the guide RNA is ineffective. On the other hand, if no 6-TG resistant cells can be obtained by co-targeting, it would suggest that the gene of interest might be essential.We have tested this method with guide RNAs for HPRT and the non-essential AAVS1 locus in HCT116 cells, a colorectal cancer cell line with a near diploidic karyotype (16). The results showed a dramatic enrichment of AAVS1 targeting events from below detection without co-targeting to over 80% with co-targeting. Other non-essential genes such as Trex1 and Exo1 were also successfully enriched by HPRT co-targeting. The method also worked in U2OS cells, an osteosarcoma cell line with a complicated karyotype (17). Co-targeting with guide RNAs for DNA topoisomerase 2α (Top2α) gave rise to no 6-TG resistant cells, which is consistent with Top2α being essential for cell proliferation (18). On the other hand, co-targeting with some other guide RNAs gave rise to plenty of 6-TG resistant cells but no alteration to the intended sequences, suggesting that the guide RNAs were ineffective. Together, these results demonstrate that co-targeting the HPRT gene provides a simple and efficient method to enrich successfully targeted cells. It can also be easily used to evaluate the effectiveness of guide RNAs and the essentialness of target genes.     

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.     

Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants

CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.     

Crystal Structure of the Cmr2–Cmr3 Subcomplex in the CRISPR–Cas RNA Silencing Effector Complex

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci found in prokaryotes are transcribed to produce CRISPR RNAs (crRNAs) that, together with CRISPR-associated (Cas) proteins, target and degrade invading genetic materials. Cmr proteins (Cmr1–6) and crRNA form a sequence-specific RNA silencing effector complex. Here, we report the crystal structures of the Pyrococcus furiosus Cmr2–Cmr3 subcomplex bound with nucleotides (3′-AMP or ATP). The association of Cmr2 and Cmr3 forms an idiosyncratic crevasse, which binds the nucleotides. Cmr3 shares structural similarity with Cas6, which cleaves precursor crRNA for maturation, suggesting the divergent evolution of these proteins. Due to the structural resemblance, the properties of the RNA binding surface observed in Cas6 are well conserved in Cmr3, indicating the RNA binding ability of Cmr3. This surface of Cmr3 constitutes the crevasse observed in the Cmr2–Cmr3 complex. Our findings suggest that the Cmr2–Cmr3 complex uses the crevasse to bind crRNA and/or substrate RNA during the reaction.     

Expansion of CRISPR targeting sites in Bombyx mori

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.     

Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10⁹ cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.     

Landscape of target:guide homology effects on Cas9-mediated cleavage

To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in ‘seed’ sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13–18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9–gRNA interaction and cleavage beyond the general paradigm of site determination based on the ‘seed’ sequence and PAM.     

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5′ ends (8-nt repeat-derived 5′ tag sequences) but heterogeneous 3′ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3′ end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.     

Genome Editing with CRISPR-Cas9:Can It Get Any Better?

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences.Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient,relying only on the design of a synthetic single guide RNA(sgRNA) and its co-expression with Cas9.Here,we review the progress in the design of sgRNA from the original dual RNA guide for S.pyogenes and Staphylococcus aureus Cas9(SpCas9 and SaCas9).New assays for genome-wide identification of offtargets have provided important insights into the issue of cleavage specificity in vivo.At the same time,the on-target activity of thousands of guides has been determined.These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences.It appears that for most basic research applications,cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions.Moreover,recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing.Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA,several variants of SpCas9 have recently been engineered,either with novel protospacer adjacent motifs(PAMs) or with drastically reduced off-targets.Novel Cas9 and Cas9-like proteins called Cpf 1 have also been characterized from other bacteria and will benefit from die insights obtained from SpCas9.Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.