| In recent years,the novel gene editing technology represented by CRISPR/Cas9(clustered regularly interspaced short palindromic repeats)system can effectively introduce DNA double strand break(DSB)at genomic target sites,thus effectively mediates downstream Knock out(KO)and Knock in(KI)efficiency.At present,KI has been widely used in gene function research,gene modification animal production,animal breeding and other fields.However,KI mediated by homologous directed repair(HDR)is still inefficient.KI operation plays a very important role in accurate genome modification.Specific phenotypes can be assigned to species through specific genotype modification.Therefore,how to improve KI efficiency is still worth further study.This study attempts to screen small molecule compounds that improve HDR efficiency,so as to provide a convenient scheme for efficient KI operation on cells.With 293 T cells as the research object,a variety of small molecule compounds capable of improving CRISPR/Cas9 were screened from 182 small molecule compounds by means of CRISPR/cas9-mediated homologous directed repair(HDR).We first successfully constructed the CRISPR/Cas9 system and the EGFP fluorescence reporting vector based on the ACTB and GAPDH gene loci of 293 T cells,so that the small molecule compounds promoting the efficiency of HDR can be visually and conveniently screened through the efficiency of fluorescence repair.We screened 88 small molecule compounds at the ACTB locus and 63 small molecule compounds at the GAPDH locus to improve HDR efficiency,respectively.Among them,29 small molecule compounds had a synergistic effect on the two gene loci.In particular,compounds Peficitinib and 4sc-202 had the most significant effect on HDR efficiency.We found that 5 μM Peficitinib could improve the HDR efficiency by 2.2 times compared with the control group.1 μM of 4sc-202 can increase the efficiency of HDR by2.5 times.Combined use of Peficitinib and 4sc-202 was not effective.The cytotoxicity of the two small molecule compounds was kept at normal level by CCK8.We further explored the mechanism of action of compound Peficitinib and 4SC-202 in enhancing the homologous directed repair mediated by CRISPR/Cas9.After treatment with Peficitinib and 4sc-202,the number of cells distributed in G0/G1 phase and S phase decreased,while the number of cells distributed in G2/M phase increased by more than one times significantly,indicating that the two compounds regulated the Cell cycle to prolong the stay in G2/M phase.After treatment of 293 T cells with Peficitinib and 4sc-202,the expression of hdr-related factors and NHEJ related factors were up-regulated.Since4SC-202 is a Histone deacetylase(HDAC)inhibitor,we suspect that inhibition of HDAC may promote the acetylation of chromatin,thereby maintaining the open state of chromatin and creating favorable conditions for the improvement of gene editing efficiency.Therefore,we inhibited HDAC levels in 293 T cells with Small interfering RNA(si RNA).The results showed that the down-regulation of HDAC1 and HDAC2 gene expression could promote the efficiency of CRISPR/ cas9-mediated HDR,and the down-regulation of HDAC3 gene expression could inhibit the efficiency of genomic HDR.Therefore,we believe that the improvement of HDR efficiency by HDAC inhibitor 4sc-202 May be related to the change of chromatin state and the regulation of cell cycle.The improvement of HDR efficiency by the JAK inhibitor Peficitini may be related to blocking the JAK/STAT signaling pathway and regulating the cell cycle. |
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