Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.
Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.
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DOI：
10.3390/genes11121501
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发表时间：
2020-12-13
期刊：
影响因子：
3.5
通讯作者：
Larochelle A
中科院分区：
文献类型：
--
作者：
Smith RH;Chen YC;Seifuddin F;Hupalo D;Alba C;Reger R;Tian X;Araki D;Dalgard CL;Childs RW;Pirooznia M;Larochelle A
CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR相关蛋白9）介导的基因组编辑在治疗人类遗传疾病方面具有巨大的潜力。然而，Cas9脱靶活性的可能性仍然是一个关注点。为了使用临床相关的靶细胞解决这个问题，我们将Cas9核糖核蛋白（RNP）复合物（分别靶向两个不同的基因组位点，2号染色体上的CXCR4位点和19号染色体上的AAVS1位点）电穿孔导入人动员外周血来源的造血干细胞和祖细胞（HSPCs），并通过单细胞来源的HSPC克隆的全基因组测序（WGS）以无偏的全基因组方式评估体细胞突变的获得情况。生物信息学分析鉴定出在Cas9处理的和未用Cas9处理的对照HSPC克隆中分布的超过20,000个体细胞变异（插入缺失、单核苷酸变异和结构变异）。统计分析显示，样本之间新的非靶向插入缺失的数量没有显著差异。此外，数据分析表明，在623个预测的脱靶位点没有Cas9介导的插入缺失形成的证据。与基线相比，Cas9 RNP接受样本组中新的单核苷酸变异的中位数略有升高，但未达到统计学意义。结构变异很少见，并且与Cas9介导的基因编辑过程没有明显的因果关系。我们发现，在Cas9 RNP编辑的人HSPC克隆中观察到的总体体细胞突变负荷与自然发生的背景遗传异质性水平没有区别。
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.
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影响因子：
48
作者：
Anderson KR;Haeussler M;Watanabe C;Janakiraman V;Lund J;Modrusan Z;Stinson J;Bei Q;Buechler A;Yu C;Thamminana SR;Tam L;Sowick MA;Alcantar T;O'Neil N;Li J;Ta L;Lima L;Roose-Girma M;Rairdan X;Durinck S;Warming S
通讯作者：
Warming S
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
14.8
作者：
Hu J;Meyers RM;Dong J;Panchakshari RA;Alt FW;Frock RL
通讯作者：
Frock RL
影响因子：
48
作者：
Crosetto, Nicola;Mitra, Abhishek;Silva, Maria Joao;Bienko, Magda;Dojer, Norbert;Wang, Qi;Karaca, Elif;Chiarle, Roberto;Skrzypczak, Magdalena;Ginalski, Krzysztof;Pasero, Philippe;Rowicka, Maga;Dikic, Ivan
通讯作者：
Dikic, Ivan
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH