Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing
Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing
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DOI：
10.1073/pnas.1818461116
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发表时间：
2019-10-15
影响因子：
11.1
通讯作者：
Chakraborty, Debojyoti
中科院分区：
文献类型：
--
作者：
Acharya, Sundaram;Mishra, Arpit;Chakraborty, Debojyoti
使用CRISPR/Cas9系统的基因组编辑已被用于在生物体的DNA中进行精确的可遗传变化。尽管广泛使用的化脓性链球菌Cas9(SpCas9)及其工程变异体已经有效地用于跨不同平台的众多基因编辑应用，但人们仍然担心它们可能偏离整个基因组的多个基因位点。在这里，我们报告了新氏弗朗西斯菌Cas9(FnCas9)显示出与其预期目标结合的非常高的特异性，而与非靶基因的结合可以忽略不计。当sgRNA：DNA异源双链中存在与靶标单导RNA(SgRNA)不匹配时，其与DNA的最小结合亲和力决定了其特异性。与SpCas9相比，FnCas9产生交错切割、更高的同源定向修复率和非常低的非特异性基因组编辑。我们证明了FnCas9介导的患者诱导的多能干细胞中镰状细胞突变的纠正，并提出它可以用于对各种遗传疾病的精确治疗性基因组编辑。
Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.