Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an \"enhanced specificity\" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.

A series of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiencies than SpCas9. We also compared the effects of spacer lengths of single-guide RNAs (sgRNAs; 18-21 nt for SpCas9 and 19-23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer length for a particular sgRNA was 18-21 nt for SpCas9 and 21-22 nt for SaCas9. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), indicating a characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or homology-directed repair (HDR)-mediated adeno-associated virus serotype 6 (AAV6) donor knock-in. Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.

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Base editors that do not require double-stranded DNA cleavage or homology-directed repair enable higher efficiency and cleaner substitution of targeted single nucleotides in genomic DNA than conventional approaches. However, their broad applications are limited within the editing window of several base pairs from the canonical NGG protospacer adjacent motif (PAM) sequence. In this study, we fused the D10A nickase of several Streptococcus pyogenes Cas9 (SpCas9) variants with Petromyzon marinus cytidine deaminase 1 (PmCDA1) and uracil DNA glycosylase inhibitor (UGI) and developed two new effective PmCDA1-based cytosine base editors (pBEs), SpCas9 nickase (SpCas9n)-pBE and VQR nickase (VQRn)-pBE, which expanded the scope of genome targeting for cytosine-to-thymine (C-to-T) substitutions in rice. Four of six and 12 of 18 target sites selected randomly in SpCas9n-pBE and VQRn-pBE, respectively were base edited with frequencies of 4-90% in T0 plants. The effective deaminase window typically spanned positions 1-7 within the protospacer and the single target C showed the maximum C-to-T frequency at or near position 3, counting the end distal to PAM as position 1. In addition, the modified single guide RNA (sgRNA) improved the base editing efficiencies of VQRn-pBE with 1.3- to 7.6-fold increases compared with the native sgRNA, and targets that could not be mutated using the native sgRNA were edited successfully using the modified sgRNA. These newly developed base editors can be used to realize C-to-T substitutions and may become powerful tools for both basic scientific research and crop breeding in rice.

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.

Despite the great achievements in genome editing, accurately detecting mutations induced by sequence-specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome-edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA-free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high-fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low-cost and widely applicable for rapid detection of genome-edited mutation in plants.