Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.
Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.
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DOI：
10.1016/j.stem.2014.10.004
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发表时间：
2014-11-06
期刊：
影响因子：
23.9
通讯作者：
Cowan, Chad A.
中科院分区：
文献类型：
--
作者：
Mandal, Pankaj K.;Ferreira, Leonardo M. R.;Collins, Ryan;Meissner, Torsten B.;Boutwell, Christian L.;Friesen, Max;Vrbanac, Vladimir;Garrison, Brian S.;Stortchevoi, Alexei;Bryder, David;Musunuru, Kiran;Brand, Harrison;Tager, Andrew M.;Allen, Todd M.;Talkowski, Michael E.;Rossi, Derrick J.;Cowan, Chad A.
通过CRISPR/Cas9进行基因组编辑凭借其有效性和易用性迅速成为首选工具。然而，CRISPR/Cas9介导的基因组编辑在临床相关的人体细胞中仍然未经测试。在这里，我们报告了CRISPR/Cas9在原代人CD 4 + T细胞和CD 34+造血干细胞和祖细胞（HSPC）中靶向两种临床相关基因B2 M和CCR 5的情况。使用单一RNA指导物导致HSPC中而不是T细胞中的高效诱变。双指导方法改善了两种细胞类型中的基因缺失功效。经过CRISPR/Cas9基因组编辑的HSPC保留了多谱系潜力。我们通过HSPC中的靶捕获测序检查了预测的在靶和脱靶突变，并仅在一个位点观察到低水平的脱靶突变。这些结果表明，CRISPR/Cas9可以以最小的脱靶诱变有效地消除HSPC中的基因，这可能对基于造血细胞的治疗具有广泛的适用性。
Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
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