Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Stem Cell Reports. 2021 Apr 13;16(4):985-996. doi: 10.1016/j.stemcr.2021.02.013. Epub 2021 Mar 11.
Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.
结合 CRISPR-Cas9 技术和单链寡脱氧核苷酸（ssODN），可以将特定的单核苷酸改变引入诱导多能干细胞（iPSC）中的靶向基因组位点；然而，与删除诱导相比，ssODN 敲入频率较低。尽管已经报道了几种 Cas9 转导方法，但 CRISPR-Cas9 核酸酶在哺乳动物细胞中的生化行为仍有待探索。在这里，我们研究了影响 Cas9 体外切割活性的内在细胞因素。我们发现，细胞内 RNA 而不是 DNA 或蛋白质部分会抑制 Cas9 与单指导 RNA（sgRNA）结合，并降低酶活性。为了防止这种情况，在将 Cas9 和 sgRNA 递送到细胞之前进行预复合可以导致比 Cas9 过表达方法更高的基因组编辑活性。通过优化预复合核糖核蛋白和 ssODN 的电穿孔参数，我们实现了高达 70%的单核苷酸校正效率和高达 40%的loxP 插入效率。最后，我们可以用 HLA-C2 等位基因替换 HLA-C1 等位基因，生成组织相容性白细胞抗原定制编辑的 iPSC。
