PDF(Pubmed)
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.
摘要：
一系列成簇的规则间隔短回文重复(CRISPR)-CRISPR相关蛋白9(Cas9)系统已被工程改造用于基因组编辑。最广泛使用的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,目前仍缺乏对其详细基因编辑结果的比较.通过表征人类诱导多能干细胞（iPSCs）和K562细胞中11个位点的编辑结果，我们发现SaCas9可以比SpCas9更有效地编辑基因组。我们还比较了单向导RNA(sgRNA，对于SpCas9为18-21nt，对于SaCas9为19-23nt），并发现SpCas9和SaCas9的最佳间隔长度分别为20nt和21nt。然而,对于SpCas9和SaCas9,特定引导RNA的最佳间隔区大小分别为18-21nt或21-22nt。此外，SpCas9在前间隔区相邻基序（PAM）上游的第四个核苷酸处非同源末端连接（NHEJ）+1插入方面比SaCas9表现出更大的偏差，交错切口的特征。因此，使用SaCas9进行编辑导致NHEJ介导的双链寡脱氧核苷酸（dsODN）插入或腺相关病毒血清型6（AAV6）供体介导的同源定向修复（HDR）的敲入效率更高。最后,GUIDE-seq分析显示,与SpCas9相比,SaCas9表现出显著降低的脱靶效应。我们的工作表明SaCas9比SpCas9在基于转基因整合的治疗性基因编辑中的优异性能，以及鉴定最佳间隔区长度以实现所需编辑结果的必要性。
