体外验证SSA报告载体系统对CRISPR/Cas9的切割效率

Verification of cleavage efficiency of SSA reporter system using CRISPR/Cas9 in vitro

目的根据单链退火(single strand annealing, SSA)修复途径原理,构建萤火虫荧光素酶荧光报告基因系统,体外检测SSA报告载体对CRISPR/Cas9的切割效率及gRNA的特异性和剪切活性。方法基于SSA修复DNA双链断裂损伤需要断裂末端包含一段同源重复序列的原理,构建4个萤火虫萤光素酶luciferase基因报告质粒,其中luciferase基因被分成前后两个片段;PCR先扩增luciferase基因319~1 653 bp片段并连接至巨细胞病毒(cytomegalovirus, CMV)启动子表达质粒,随后再分别连接luciferase基因不同长度前段序列,分别为1~579 bp、1~858 bp、1~1 188 bp和1~1 317 bp,每个片段中均含有一段重复序列,并且被终止密码子提前终止为无活性的luciferase基因;设计了261、540、870和900 bp等4个同源臂,将特异性的gRNA连入两段序列中,并加入有活性的Cas9/gRNA,通过检测荧光活性来检测gRNA的特异性和剪切活性。结果成功构建了4个荧光报告质粒pLuc-N1-1~4;用海参荧光素酶作为内参,在293T细胞转染4个荧光报告质粒,在24 h时检测4个质粒的相对荧光强度,均无荧光活性物质产生;在荧光报告质粒中加入特异性的靶点序列,并表达Cas9/gRNA,这些报告基因具有显著荧光强度升高(P<0.000 1),且不受同源臂长度影响。结论成功构建的基于luciferase基因的SSA报告系统,可提供一种简单而快速方法来评估和比较gRNA在CRISPR/Cas9系统引入的indel突变诱导效率,为选择最有效gRNA减少不确定性,大大扩展CRISPR/Cas9介导的模式动物基因工程的实际应用提供可能性。

Objective According to the principle of single strand annealing(SSA) repair pathway, a fluorescent reporter system using firefly luciferase was developed to detect the cleavage efficiency of SSA reporter vector using CRISPR/Cas9 and to evaluate the specificity and splicing activity of gRNA in vitro. Methods Four reporter plasmids containing firefly luciferase genes were constructed based on the principle that SSA mediates double-strand DNA breaks repairing between homologous repeated sequences. The luciferase gene was divided into two fragments. The fragment(319-1653 bp) was amplified by PCR and ligated into the CMV promoter expression plasmid. And then the other segments of luciferase with different lengths(1-579 bp, 1~858 bp, 1-1188 bp and 1-1317 bp) were cloned into the above plasmid. Each fragment contained a repeated sequence and the stop codon was inserted into the gene, resulting in production of inactive luciferase. Four homologous arms(261 bp, 540 bp, 870 bp and 900 bp) were designed. The specific gRNA was connected into the two segments, and the active Cas9/gRNA was added to detect the specificity and splicing activity of gRNA by detecting the fluorescence activity. Results Four fluorescent reporter plasmids pLuc-N1-1,2,3,4 were successfully constructed. Four fluorescent reporter plasmids were transfected into 293 T cells, and sea cucumber luciferase was used as an internal reference. The relative fluorescence intensity of the four plasmids was detected at 24 h, and no fluorescent substance was produced. The fluorescence intensity of these reporter genes significantly increased(P<0.000 1) by adding specific target sequence and expressing Cas9/gRNA, and was not affected by the length of homologous arm. Conclusion SSA reporter system based on luciferase was successfully constructed, which can provide a simple and rapid method to evaluate the efficiency of indel mutation introduced by gRNA in CRISPR/Cas9 system, and can promote selection of the most effective gRNA, and greatly expand the practical application of CRISPR/Cas9-mediated model animal genetic engineering.