SpCas9 Variants Research Articles

Structural and Dynamics Studies of the Spcas9 Variant Provide Insights into the Regulatory Role of the REC1 Domain

CRISPR-Cas9 from Streptococcus pyogenes has been harnessed as a powerful biotechnological tool for DNA sequence modification. The gRNA binding the recognition lobe (REC lobe) of Cas9 is the initial step of Cas9-mediated DNA cleavage and is a prerequisite to trigger the subsequent conformational transition of Cas9. The functions of REC1–3 domains have been summarized as “sense” nucleic acids, “regulate” the HNH conformational transition, and finally “lock” the HNH domain at the cleavage site. Therefore, understanding the dynamic mechanism of the REC lobe is important to design and engineer better Cas9 enzymes. Here, we solved the apo form structure of a SpCas9 variant (xCas9 P411T) that harbors seven mutations in the SpCas9 REC region and one mutation in the C-terminal domain. This structure reveals that the mutations in the REC lobe resulted in a distinct conformation of the REC1 domain. Together with the biochemical results, we hypothesized that the dynamic properties of the REC1 domain are associated with Cas9 PAM specificity. Furthermore, using molecular dynamics simulations to model the conformational activation process of xCas9 P411T, we found that REC1 acts as a hub that modulates multiple domain motion during Cas9 activation and target site binding. These results lay the foundation for engineering an improved CRISPR-Cas9 system.

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs). Horticultural crops provide energy and health-keeping nutrients to humankind. Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops. Here, we describe the CRISPR/Cas9 system construction, its optimization, including sgRNA promoter, sgRNA design, Cas9 protein promoter, SpCas9 variants and orthologs, and vector delivery methods. We also summarized the application of this technology in horticultural plants for stress responses enhancement, fruit quality improvement, and cultivation traits modification. This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo.

CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively.

Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9.

The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.

BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

Editing Properties of Base Editors with SpCas9-NG in Discarded Human Tripronuclear Zygotes.

DNA base editors, comprising nucleotide deaminases and catalytically impaired Cas9 nickase, have been widely used in various organisms for the efficient creation of point mutations, providing researchers with powerful tools in precise genome editing. However, they have been limited by the scope of the editing. The discovery and engineering of various CRISPR-Cas systems, especially SpCas9 variants xCas9, Cas9-NG, and Cas9-SpRY, have diversified the range of targetable DNA sequences and expanded the targeting scope of genomic base editing. To understand the editing properties comprehensively, we conducted an analysis of the editing properties of adenine base editors and cytosine base editors with xCas9, Cas9-NG, and Cas9-SpRY at endogenous sites with NGN protospacer adjacent motifs (PAM). Then, human genetic disease-associated DNA point mutations were installed at a single site or at dual sites with NGH PAM using base editors with SpCas9-NG (ABEmax-NG and Anc-BE4max-NG [BEs-NG]) in cultured human cell lines. Finally, the editing properties of BEs-NG in discarded human tripronuclear embryos were characterized. This study investigated the editing properties of DNA base editors with a relaxed PAM requirement and demonstrated the potential of BEs-NG in human genetic disease-related research and treatment.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

CABE-RY: A PAM-flexible dual-mutation base editor for reliable modeling of multi-nucleotide variants

Multi-nucleotide variants (MNVs) represent an important type of genetic variation and have biological and clinical significance. To simulate MNVs, we designed four dual-mutation base editors combining hA3A(Y130F), TadA8e(V106W), and protospacer adjacent motif (PAM)-flexible SpRY and selected cytosine and adenine base editor-SpRY (CABE-RY), which had the best editing performance, for further study. Characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors. Thus, we have established a versatile tool to efficiently simulate MNVs over the genome, which could be very useful for functional studies on MNVs in humans.

Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR–Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR–Cas9 editing, raising an urgent issue to be addressed.

Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

The application of genome editing technology in fish.

The advent and development of genome editing technology has opened up the possibility of directly targeting and modifying genomic sequences in the field of life sciences with rapid developments occurring in the last decade. As a powerful tool to decipher genome data at the molecular biology level, genome editing technology has made important contributions to elucidating many biological problems. Currently, the three most widely used genome editing technologies include: zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR). Researchers are still striving to create simpler, more efficient, and accurate techniques, such as engineered base editors and new CRISPR/Cas systems, to improve editing efficiency and reduce off-target rate, as well as a near-PAMless SpCas9 variants to expand the scope of genome editing. As one of the important animal protein sources, fish has significant economic value in aquaculture. In addition, fish is indispensable for research as it serves as the evolutionary link between invertebrates and higher vertebrates. Consequently, genome editing technologies were applied extensively in various fish species for basic functional studies as well as applied research in aquaculture. In this review, we focus on the application of genome editing technologies in fish species detailing growth, gender, and pigmentation traits. In addition, we have focused on the construction of a zebrafish (Danio rerio) disease model and high-throughput screening of functional genes. Finally, we provide some of the future perspectives of this technology.

Reduced off-target effect of NG-BE4max by using NG-HiFi system

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State.

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. However, due to difficulty in determining active CRISPR-Cas9 structure in DNA cleavage state by X-ray and cryo-EM, it remains uncertain how the HNH and RuvC nuclease domains in CRISPR-Cas9 split the DNA phosphodiester bonds with metal ions and water molecules. Therefore, based on one-and two-metal-ion mechanisms, homology modeling and molecular dynamics simulation (MD) are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9-sgRNA-DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg2+ ions and water. Among them, residues D10, E762, H983 and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provides new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state, and offers useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

Massively Parallel Kinetic Profiling of Natural and Engineered CRISPR Nucleases

Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than natural SpCas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq—nuclease digestion and deep sequencing—a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered SpCas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to natural SpCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of SpCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.

Engineered dual selection for directed evolution of SpCas9 PAM specificity

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

BackgroundPlant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome.ResultsIn this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines.ConclusionsThe broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.