Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.
Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.
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DOI：
10.1016/j.stemcr.2021.02.013
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发表时间：
2021-04-13
影响因子：
5.9
通讯作者：
Hotta A
中科院分区：
文献类型：
--
作者：
Kagita A;Lung MSY;Xu H;Kita Y;Sasakawa N;Iguchi T;Ono M;Wang XH;Gee P;Hotta A
结合CRISPR-Cas9技术和单链寡脱氧核苷酸（ssODN），可以将特异性单核苷酸改变引入诱导多能干细胞（iPSC）的靶基因组位点;然而，与缺失诱导相比，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物化学行为还有待探索。在这里，我们研究了影响Cas9体外切割活性的内在细胞因子。我们发现细胞内RNA而不是DNA或蛋白质部分抑制Cas9与单向导RNA（sgRNA）结合并降低酶活性。为了防止这种情况，与Cas9过表达方法相比，在递送到细胞中之前预复合Cas9和sgRNA可以导致更高的基因组编辑活性。通过优化预复合的核糖核蛋白和ssODN的电穿孔参数，我们实现了高达70%的单核苷酸校正和loxP插入高达40%的效率。最后，我们可以用C2等位基因取代HLA-C1等位基因，以产生组织相容性白细胞抗原定制编辑的iPSCs。细胞内RNA，而不是DNA或蛋白质，抑制Cas9与sgRNA结合预复合Cas9和sgRNA优于Cas9过表达方法优化的电穿孔允许在没有抗生素的情况下，在iPSC loxP插入和双等位基因单核苷酸校正中高速率的ssODN供体基因组编辑。发现细胞内RNA而不是DNA或蛋白质部分抑制Cas9与sgRNA结合并降低酶活性。为了避免这种情况，预复合Cas9和sgRNA可以导致比Cas9过表达更高的基因组编辑活性。通过优化电穿孔条件，双等位基因单核苷酸改变和loxP插入在iPSC中是可行的，而无需抗生素选择。
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. Intracellular RNA, but not DNA or protein, inhibits Cas9 from binding to sgRNA Precomplexing Cas9 and sgRNA is preferable to Cas9 overexpression approaches Optimized electroporation permits high rates of ssODN-donor genome editing in iPSCs loxP insertions and biallelic single-nucleotide corrections without antibiotics Hotta et al. found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to sgRNA and reduces the enzymatic activity. To avoid this, precomplexing Cas9 and sgRNA could lead to higher genome editing activity than Cas9 overexpression. By optimizing electroporation conditions, biallelic single-nucleotide alterations and loxP insertion were feasible without antibiotic selection in iPSCs.
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