Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions.
Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions.
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DOI：
10.1371/journal.pone.0144970
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Bleris L
中科院分区：
文献类型：
--
作者：
Li Y;Mendiratta S;Ehrhardt K;Kashyap N;White MA;Bleris L
CRISPR/Cas9是一种rna引导的基因组靶向和工程技术。Cas9蛋白的一个急性DNA结合约束是原间隔邻近基序（Protospacer邻基序，PAM）。在这里，我们证明了PAM需求可以被用来特异性地靶向单核苷酸杂合突变，而对野生型等位基因没有产生异常影响。具体来说，我们针对结直肠癌细胞中KRAS的杂合G13A激活突变，我们显示了对MEK小分子抑制剂的耐药性逆转。我们的研究通过使用gRNA引导Cas9到所需的原间隔器邻近基序，引入了基因组编辑和治疗靶向的新范式。
CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.