Abstract
The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 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 Streptococcus pyogenes 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 IGF2 SNP editing by silencing the LIG4 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 IGF2 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 IGF2 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.
Highlights
The CRISPR/Cas9 technology (Jinek et al, 2012; Mali et al, 2013) has been widely used for genome editing in various cell types and organisms since its advent
The DsRed gene was used as the transfection marker, and the interrupted eGFP gene was used as the reporter, which was designed to be repaired accurately by single strand annealing (SSA) when targeted by the sgRNA/Cas9 complex (Xu et al, 2015a)
As limited clones were sequenced by Sanger sequencing, which may not reveal the real difference between the two experiment groups, we further conducted the Deep-sequencing analysis and the results demonstrated a significantly higher homology-directed repair (HDR)-based editing efficiency (16.38%) for the sgRNA-shRNA/StCas9 as S. thermophilus (StCas9) group (Figure 4I, ∗P < 0.05)
Summary
The CRISPR/Cas technology (Jinek et al, 2012; Mali et al, 2013) has been widely used for genome editing in various cell types and organisms since its advent. The Cas endonuclease is directed by an artificial singleguide RNA (sgRNA or gRNA) to recognize the target DNA sequence with given protospacer adjacent motif (PAM) by base pairing (Jinek et al, 2012). The DNA double-strand breaks (DSBs) induced by the endonucleases can be repaired by two main mechanisms, the error-prone non-homologous end joining (NHEJ) and the donor-dependent homology-directed repair (HDR). The HDR pathway can result in desired genome editing events by targeted recombination of designed homologous DNA template donors (Salsman and Dellaire, 2017)
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