Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1)to nick the target DNA strand

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.

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of application.Recently,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human cells.In this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable transformation.Moreover,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants.Our results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice lines.Collectively,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing

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.

识别NG PAM序列的Cas9变异体编辑玉米ZmSCD基因及其效率测试

为了探究评估SpCas9-NG在玉米基因组编辑中的应用潜力,以叶绿素合成关键基因ZmSCD作为编辑对象,该基因缺失会使苗出现白化现象,以此直观评估编辑效率。依据SpCas9和SpCas9-NG分别识别的5′-NGG-3′和5′-NG-3′PAM序列的规则,在ZmSCD的第2和第3外显子上设计了靶点后,成功将靶点序列构建至SpCas9及SpCas9-NG敲除载体上,再利用农杆菌介导的遗传转化方法将敲除载体导入玉米自交系KN5585中,将愈伤组织培养至分化出叶片组织时,统计白化苗率,以此得出不同编辑器的编辑效率,并对白化苗提取基因组后进行测序。通过3轮遗传转化SpCas9分别获得76,125,28个愈伤组织,SpCas9-NG获得100,69,30个愈伤组织。结果显示,SpCas9的3轮遗传转化的基因编辑效率分别为14.47%,13.60%,10.71%,SpCas9-NG编辑效率分别为12.00%,10.14%,13.33%;对其白化苗测序结果显示,均在靶点附近获得重叠峰。结果表明,SpCas9-NG在玉米中的编辑效率与传统SpCas9相似,显示出同样的基因编辑能力;相比之下,SpCas9-NG具备更宽泛的PAM序列适应性,这意味着它的靶点设计能力更为灵活,允许在玉米基因组中实现更加精准和多样化的编辑。

CRISPR/cas系统及其在家禽上的应用研究进展

CRISPR/cas是一种获得性免疫防御系统,广泛存在于细菌和古细菌中。Strep tococcus pyogenes cas9(Spcas9)作为目前研究最多、最清楚的效应蛋白,因其酶活性高和靶点广而被应用于生命科学各个领域,但Spcas9分子量大、脱靶率高等缺点在一定程度上限制了其应用。随着CRISPR系统技术被深入开发,一些新效应蛋白被发现,本文就CRISPR/cas系统分类、原理、新效应蛋白发现及cas9(S.Pyogenes)多种变构体技术开发在及家禽上的应用进行综述,为相关领域的研究提供参考。

Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences

CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.

CRISPR-Cas nucleases and base editors for plant genome editing

Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.