Research And Optimization Of C18 Homology Template-mediated Gene Knock-in

Gene knock-in technology mainly uses the homologous sequences that humans rely on to introduce gene repair,and uses the cell's own DNA homologous recombination repair mechanism to achieve the site-specific insertion of foreign genes.The new generation of CRISPR/Cas9 gene editing technology is a kind of gene knock-in,knock-out and modification technology with simple operation,better precision,higher efficiency and better safety.At present,it mainly mediates gene targeting through the following 5 methods.Integration,namely homologous recombination(HR),microhomology-mediated end joining(MMEJ),non-homologous end joining(NHEJ),homology-mediated end joining(HMEJ)and single-stranded oligodeoxynucleotides,(ss ODNs).In this study,Spacer18(C18)can be connected to the nucleotide chain to develop a new method of C18 homology template-mediated gene knock-in.By designing a single-stranded nucleotide primer containing C18,the knock-in gene is used as Template,using PCR to generate C18 homologous template with sticky ends at both ends.Based on this,six gene knock-in methods mediated by C18 homology template,HR,MMEJ,NHEJ,HMEJ and ss ODN were compared for knock-in efficiency and accuracy.At the same time,the C18 homology template method was used to optimize the homology arm length and plasmid transfection dose,and improve the efficiency and accuracy of gene knock-in.The test process and the main results obtained are as follows:1.Construction of multiple homologous targeting vectors.A C18 homologous template primer was designed.The primer had a 40 bp homologous sequence at the 5' end and a knock-in fragment at the 3' end.The C18 homologous template was successfully obtained by PCR.GFP is connected together through C18.At the same time,the HR vector with 800 bp homology arm and no sg RNA recognition site,the HMEJ vector with 800 bp homology arm and sg RNA recognition site,the NHEJ vector with sg RNA recognition site but no homology arm,containing MMEJ vector with 40 bp homology arm and containing sg RNA recognition site.The company was commissioned to synthesize the ss ODN with a homology arm length of 40 bp.2.Comparison of the efficiency of different gene knock-in methods.The above homologous targeting vector,sg RNA vector and Cas9 vector were co-transfected into mouse myoblasts(C2C12).After screening with puromycin and blastin,the efficiency of gene knock-in was detected by flow cytometry.The results showed that the efficiency of C18 homologous template-mediated gene knock-in was 1.15%,significantly higher than MMEJ(0.3%),NHEJ(0.17%)-mediated gene knock-in efficiency,but significantly lower than HMEJ(1.81%)and HR(3.17%)-mediated gene knock-in efficiency.3.Comparison of the accuracy of different gene knock-in methods.Design two pairs of primers based on the front end of the knock-in sequence and the entire sequence,and perform real-time quantitative PCR.By absolute quantitative analysis of its copy number,the accuracy of gene knock-in was identified.It was found that based on the gene knock-in method mediated by the C18 homology template,there was no significant difference in the copy numbers of the inserted sequences corresponding to the two pairs of primers,indicating that the C18 homology template-mediated gene knock-in method has the highest accuracy.4.C18 homology template knock-in method for target gene locus stability analysis.Rosa26 and GAPDH were selected as the target genes,and used the C18 homology template with a homology arm of 40 bp to knock the T2A-Dsred-Poly A fragment(973 bp)and T2A-GFP(824 bp)into these two targets.The results of flow cytometry showed that the efficiency of Dsred knock-in at Rosa26 site was 1.12%,and the efficiency of GFP knock-in at GAPDH site was 1.15%.When the lengths of the inserts were not significantly different and were below 1 kb,The C18 homology template-mediated gene knock-in method can produce efficient and non-different knock-in at these two gene loci.5.Optimization of transfection dosage of cell liposome transfection method.Using 5 ?L Lipo6000 ?,the GFP-expressing Cas9-GFP plasmid was transfected into mouse myoblasts(C2C12)cultured in 6-well plates at the doses of 0.5,1.0,2.0,and 5.0 ?g / m L,respectively.The transfection efficiency was 1.90%,4.23%,5.47%,and 5.20%,respectively,and the highest transfection efficiency was determined when the transfection concentration was 2.0 ?g / m L.6.Optimization of the length of homology arm of C18 homology template.The first intron region of the Rosa26 gene was used as the target site,the sg RNA-2 with the highest editing efficiency was selected,and the T2A-Dsred-Poly A was used as the knock-in fragment.Primers,PCR amplification to obtain C18 homologous templates with different homology arm lengths,and then transfected cells with Cas9 plasmid and sg RNA-2 plasmid,the gene knock-in efficiency was detected as 0.48%,1.01%,1.12%,0.39%.The optimal length of the homology arm is 40 bp,at which time the knock-in efficiency is the highest.In summary,this study constructed a new method of C18 homology template-mediated gene knock-in,which has high knock-in efficiency and accuracy,and has stable knock-in efficiency at different target gene loci.The method provides new ideas for gene integration methods and can be applied to construct animal models of gene knock-in.