State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA 92350, USA.
Nucleic Acids Res. 2021 Jan 25;49(2):969-985. doi: 10.1093/nar/gkaa1251.
Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.
CRISPR 基因敲除编辑谱的研究有助于提高编辑结果的精确性。然而，特别是对于同源定向修复（HDR），在临床相关细胞（如人类 iPSC 和原代 T 细胞）中，其编辑动力学和模式仍知之甚少。在这里，我们研究了 Cas9 指导 RNA 核糖核蛋白（RNP）递送后，在四种细胞类型中，有无腺相关病毒血清型 6（AAV6）作为 HDR 供体时的编辑动力学和 DNA 修复谱。我们发现编辑谱在细胞系之间存在明显差异。我们还揭示了 AAV6 供体模板介导的 HDR 动力学。T50（达到最大编辑频率一半的时间）的定量表明，短插入缺失（尤其是 +A/T）比更长的缺失（>2 bp）发生得更快，而 HDR 的动力学介于非同源末端连接（NHEJ）和微同源介导的末端连接（MMEJ）之间。因此，AAV6 介导的 HDR 有效地与更长的 MMEJ 介导的缺失竞争，但不能与 NHEJ 介导的插入缺失竞争。值得注意的是，小分子化合物 M3814 和 Trichostatin A（TSA）的组合，能够强烈抑制主要的 NHEJ 修复，可使 HDR 效率提高 3 倍。
