Cpf1 和 Cas9核酸酶靶向EGFR-L858R突变的研究
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Q291

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Targeting EGFR-L858R mutation by Cpf1 and Cas9 nuclease
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    摘要:

    EGFR的突变是造成非小细胞肺癌的原因之一。EGFR第19个外显子的缺失和L858R (2573T>G)单核苷酸突变占所有EGFR突变的90%。选择性地使突变EGFR失活对这类突变的病人是非常有利的。这里,应用双荧光报告分析的方法分析CRISPR-Cpf1和CRISPR-Cas9系统在靶向EGFR-L858R突变的编辑效率。在EGFR-L858R突变位点的附近,有两个Cpf1前间区序列邻近基序(PAMs)——TTTN。并且,EGFR的2573T>G突变形成了一个Cas9的PAM——NGG。因此本文通过构建两条AsCpf1的gRNAs (gRNA1和gRNA2)和一条SpCas9的 gRNA (gRNA3) 在体外通过双荧光蛋白分析系统去评估SpCas9和 AsCpf1特异性靶向等位基因的能力。结果证实了AsCpf1和SpCas9 都能够特异性的编辑突变的EGFR(2573T>G)。实验结果显示Cpf1和Cas9 能够通过高特异性破坏突变癌基因,从而具有潜在的精准治疗价值。

    Abstract:

    Mutations in the EGFR kinase are a cause of non-small-cell lung cancer. Deletions in exon 19 and the L858R (2573T>G) single point mutation constitute about 90% of all EGFR mutations. Selectively inactivate only mutant, not normal allele, could benefit patients with such mutations. Here, the editing efficacy and selectivity of CRISPR-Cpf1 and -Cas9 systems on EGFR L858R mutant allele were analyzed by dual-reporter assay in vitro. Near the mutation site, there are two TTTN protospacer adjacent motifs (PAMs) for Cpf1. 2573T>G substitution also leads to occurrence of a novel NGG PAM for Cas9. Thus we designed two AsCpf1 gRNA (gRNA1 and gRNA2) and one SpCas9 gRNA (gRNA3) and evaluated their potency and allele specificity in vitro using a dual fluorescent protein-based bioassay system. As a result, both AsCpf1 and SpCas9 demonstrated robust activities to induce specific editing of only 2573T>G mutant EGFR, not wild-type sequence. Our results support the potential applicability of both Cpf1 and Cas9 in precision medicine through highly specific disruption of mutant oncogenes.

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引用本文格式: 魏恒,杨梅佳,钟坤宏,仝爱平. Cpf1 和 Cas9核酸酶靶向EGFR-L858R突变的研究[J]. 四川大学学报: 自然科学版, 2018, 55: 401.

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  • 收稿日期:2017-03-29
  • 最后修改日期:2017-05-10
  • 录用日期:2017-05-19
  • 在线发布日期: 2018-03-18
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