Cpf1和Cas9核酸酶靶向EGFR-L858R突变的研究

Targeting EGFR-L858R mutation by Cpf1 and Cas9 nuclease

表皮生长因子受体(EGFR)单核苷酸突变(2573T>G,L858R)占所有EGFR突变的90%.使突变的EGFR失活对有此突变的病人非常有利.这里,应用双荧光报告分析的方法分析规律成簇间隔短回文重复(CRISPR)系统中Cpf1和Cas9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,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).

Epidermal growth factor receptor（EGFR）single point mutation（2573T^G, L858R） 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 clustered regularly interspaced short palindromic repeats（CRISPR）Cpfl 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 Cpfl. 2573T^G substitution also leads to occurrence of a novel NGG PAM for Casg. Thus we designed two AsCpfl 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 AsCpfl and SpCas9 demonstrated robust activities to induce specific editing of only 2573T〉 G mutant EGFR.