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

IF 11.5 2区 生物学 Q1 GENETICS & HEREDITY Genomics, Proteomics & Bioinformatics Pub Date : 2023-12-01 Epub Date: 2022-12-20 DOI:10.1016/j.gpb.2022.12.003
Zhi-Xue Yang, Ya-Wen Fu, Juan-Juan Zhao, Feng Zhang, Si-Ang Li, Mei Zhao, Wei Wen, Lei Zhang, Tao Cheng, Jian-Ping Zhang, Xiao-Bing Zhang
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Abstract

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.

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与 SpCas9 相比,SaCas9 在基因组编辑中具有更高的保真度和不同的编辑结果。
目前已设计出一系列用于基因组编辑的簇状规则间隔短回文重复序列(CRISPR)-CRISPR 相关蛋白 9(Cas9)系统。使用最广泛的 Cas9 是来自化脓性链球菌的 SpCas9 和来自金黄色葡萄球菌的 SaCas9。然而,目前仍缺乏对其基因编辑结果的详细比较。通过分析人类诱导多能干细胞(iPSC)和 K562 细胞中 11 个位点的编辑结果,我们发现 SaCas9 比 SpCas9 能更有效地编辑基因组。我们还比较了单导RNA(sgRNA,SpCas9为18-21 nt,SaCas9为19-23 nt)的间隔长度,发现SpCas9和SaCas9的最佳间隔长度分别为20 nt和21 nt。然而,对于 SpCas9 和 SaCas9 而言,特定引导 RNA 的最佳间隔长度分别为 18-21 nt 或 21-22 nt。此外,与 SaCas9 相比,SpCas9 在非同源末端连接(NHEJ)+1 插入原间隔邻接基序(PAM)上游第四个核苷酸时表现出更大的偏差,这是交错切割的特征。因此,用SaCas9进行编辑可提高NHEJ介导的双链寡核苷酸(dsODN)插入或腺病毒血清型6(AAV6)供体介导的同源定向修复(HDR)的基因敲入效率。最后,GUIDE-seq分析显示,与SpCas9相比,SaCas9的脱靶效应明显降低。我们的工作表明,在基于转基因整合的治疗性基因编辑中,SaCas9的性能优于SpCas9,而且有必要确定最佳间隔长度,以实现理想的编辑效果。
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来源期刊
Genomics, Proteomics & Bioinformatics
Genomics, Proteomics & Bioinformatics Biochemistry, Genetics and Molecular Biology-Biochemistry
CiteScore
14.30
自引率
4.20%
发文量
844
审稿时长
61 days
期刊介绍: Genomics, Proteomics and Bioinformatics (GPB) is the official journal of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China. It aims to disseminate new developments in the field of omics and bioinformatics, publish high-quality discoveries quickly, and promote open access and online publication. GPB welcomes submissions in all areas of life science, biology, and biomedicine, with a focus on large data acquisition, analysis, and curation. Manuscripts covering omics and related bioinformatics topics are particularly encouraged. GPB is indexed/abstracted by PubMed/MEDLINE, PubMed Central, Scopus, BIOSIS Previews, Chemical Abstracts, CSCD, among others.
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