sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9

• Published in: Frontiers in genetics Vol. 10; p. 347
• Main Authors: Sun, Yongsen, Yan, Nana, Mu, Lu, Sun, Bing, Deng, Jingrong, Fang, Yuanyuan, Shao, Simin, Yan, Qiang, Han, Furong, Zhang, Zhiying, Xu, Kun
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 18.04.2019
• Subjects: base editing; CRISPR; Genetics; IGF2 gene; sgRNA-shRNA; SNP; StCas9; sgRNA-shRNA; CRISPR; StCas9; IGF2 gene; SNP; base editing
• ISSN: 1664-8021; 1664-8021
• DOI: 10.3389/fgene.2019.00347

The SNP within intron 3 of the porcine gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the SNP site, which we called "SNP editing." The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based SNP editing by silencing the gene, which is a key molecule of the HDR's competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases.