二代靶向测序在CRISPR/Cas9靶区筛选中的应用性研究

doi:10.11843/j.issn.0366-6964.2019.08.007

摘要/Abstract

摘要： 旨在利用二代靶向测序技术比较不同靶区对CRISPR/Cas9基因编辑结局的影响，为该技术应用过程中的靶区筛选提供技术参考。本研究进行如下试验：1）以小鼠Pyk2基因为研究对象，针对其第一外显子区设计3个靶区，通过打靶质粒构建、细胞转染及转染细胞DNA二代靶向测序等手段发现，不同靶区总体的基因编辑（Indels）效率相差较大（site1：32.0%；site2：7.9%；site3：69.5%），编辑修复的偏好性也存在较大区别；而对于同一靶区，总编辑效率在2组试验重复中较为稳定（site1：31.8%vs 32.3%；site2：7.4%vs 8.4%；site3：71.3%vs 67.8%），编辑的偏好性也相对一致；2）针对上述筛出的最佳靶区site1，通过sgRNA与Cas9体外转录、小鼠胚胎显微注射和移植及子代的基因型鉴定等手段，结果发现，上述靶区的基因编辑偏好性在基因编辑动物生产中也得到证实；3）利用上述得到的site1靶区插入一个碱基的突变小鼠，在原靶区位置设计与site1仅相差一个碱基的sgRNA。通过突变小鼠基因组PCR和Cas9酶体外切割试验发现，靶区切割位点单碱基的插入就对该靶区编辑的效率产生显著影响（49.2%vs 0%）。综上所述，靶区选择对CRISPR/Cas9基因编辑的结局影响显著，通过二代靶向测序可以有效筛选CRISPR-Cas9基因编辑靶区，并在模式动物生产过程中也获得较好的结果。此外，针对靶区切割位点的单个碱基插入对基因编辑效率影响显著。

关键词: 靶向测序, CRISPR/Cas9, 基因编辑, Pyk2

Abstract: This work aimed to compare the effects of different target regions on the editing outcomes of CRISPR/Cas9 technology by using next generation targeted sequence, and provide a technical reference for target screening during application of this technology. This work was carried out as follows:1) Mouse Pyk2 gene was used in this study, and 3 target regions were designed in the first exon of this gene, through plasmid construction, cell transfection and next generation targeted sequencing of the cell DNA, we found that the total gene editing (Indels) efficiency of different target regions was different(site1:32.0%;site2:7.9%;site3:69.5%), and the preference for gene repair were also different among different groups. For the same target region, the editing efficiency was more stable in 2 experimental replicates(31.8% vs 32.3% for site1;7.4% vs 8.4% for site2;71.3% vs 67.8% for site3), and the preference of gene editing was relatively conservative; 2) Through Pyk2 site1 sgRNA and Cas9 in vitro transcription, mouse embryos microinjection, embryo transfer and genotyping, we found that the gene editing preference of the above target region was reproducible in gene editing animals production; 3) The mutant mouse with a single base inserted in site1 target region was used, sgRNA with a different base from site1 in orginal target was desigend. For the Pyk2 site1 target region, we found that single base inserts on Cas9 cleavage site had a significant impact on the efficiency of gene editing in the target region (49.2% vs 0%) by mutated mouse genomic PCR and Cas9 enzyme digestion in vitro cleavage test. In conclusion, target selection has a significant impact on the outcome of CRISPR/Cas9 gene editing. The next generation targeted sequence can effectively screen the CRISPR/Cas9 gene editing target region, and also obtain optimal results in the model animal production. In addition, inserting single base to the target region has a significant effect on gene editing efficiency.

Key words: targeted sequence, CRISPR/Cas9, gene editing, Pyk2

中图分类号:

S813

吴海波, 边雪娇, 贾丽玲, 索伦. 二代靶向测序在CRISPR/Cas9靶区筛选中的应用性研究[J]. 畜牧兽医学报, 2019, 50(8): 1587-1595.

WU Haibo, BIAN Xuejiao, JIA Liling, SUO Lun. Screening of CRISPR/Cas9 Gene Editing Targets Using Next Generation Targeted Sequence[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1587-1595.

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