SaCas9 Requires 5-NNGRRT-3 PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5-NNGRRT-3 PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

CRISPR/Cas9介导的基因治疗在人类疾病治疗方面具有巨大的潜力。前间区序列邻近基序（PAM），即与靶序列相邻的序列，是设计CRISPR/Cas9介导的基因编辑的关键靶向元件。然而，目前很少有研究尝试直接研究人类细胞中的PAM序列。为了解决这一问题，作者开发了一种双荧光报告系统，可用于识别基因组编辑内切酶（包括Cas9）的功能性PAM。利用该系统，作者研究了不同PAM序列对SaCas9的影响，SaCas9体积小，具有可进行体内基因组编辑的优势，并且发现只有5 - NNGRRT - 3这种PAM能够在多个位点诱导足够的靶标切割。作者还通过基于整合eGFP的质粒（附加体）和染色体比较发现，SaCas9比SpCas9或FnCpf1具有更高的活性。总之，作者表明双荧光报告系统是一种识别功能性PAM以及定量比较人类细胞中具有相似或相同靶序列的不同基因组编辑内切酶效率的方法。