PDF(Pubmed)
Abstract：
The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.
摘要：
CRISPR疗法的临床成功取决于Cas蛋白的安全性和有效性。来自Francisellanovicida（FnCas9）的Cas9非常精确，对错配底物的亲和力可忽略不计，但其低细胞靶向效率限制了治疗用途。这里,我们合理地设计蛋白质以开发增强的FnCas9（enFnCas9）变体，并将其在人类基因组位点的可及性扩大约3.5倍。具有单一错配特异性的enFnCas9蛋白扩展了基于FnCas9的CRISPR诊断的目标范围以检测致病性DNA特征。它们在目标编辑效率方面优于化脓性链球菌Cas9（SpCas9）及其工程衍生物，敲入率，和脱靶特异性。enFnCas9可以与延伸的gRNA组合,用于在PAM约束的规范碱基编辑器不可访问的位点处进行稳健的碱基编辑。最后,我们证明了使用enFnCas9腺嘌呤碱基编辑器在Leber先天性黑蒙2（LCA2）患者特异性iPSC系中的RPE65突变校正，强调其治疗效用。
