Development And Application Of A Highly Enhanced Gene-editing Tools

The rapid development of gene editing technologies represented by CRISPR/Cas9 has played an important role in the construction of animal models,screening of drug targets and gene therapy.At present,CRISPR/Cas9 has been extensively optimized in both targeting range and specificity,and a variety of Cas9 variants with wider targeting range and higher specificity have been developed.However,there are relatively few studies on improving CRISPR/Cas9-mediated gene editing efficiency,and the existing studies mainly focus on improving the efficiency of gene editing by optimizing Cas9 codon and nuclear localization signal,as well as enhancing chromatin accessibility in cells.Therefore,we want to develop an efficient gene editing tools to improve gene editing efficiency by enhancing the DNA binding ability of Cas9.We firstly screened 11 non-sequence-specific DNA double-stranded binding proteins(DBDs)and found that Cas9 showed the highest gene editing efficiency when the fusion HMG-D protein originated from drosophila.Through optimizing the linker length between Cas9 and HMG-D protein,and the location or amount of HMG-D protein,we developed a highly enhanced efficiency Cas9 variant(He E-Sp Cas9).We next tested the gene editing efficiency of He E-Sp Cas9 in multiple endogenous targets.Through high throughput sequencing(HTS)analysis,we found that He ESp Cas9 increased the gene editing efficiency by 1.4-fold in 41 endogenous targets.In order to expand the targeting range of He E-Sp Cas9,we developed multiple He ESp Cas9 variants targeting “NGN” PAM or near-PAMless through fusing HMG-D protein,these variants significantly improved the gene editing efficiency up to 1.3-fold at multiple endogenous targets.Meanwhile,we also developed enhanced transcriptional activation tools(d He E-Sp Cas9-VPR)and enhanced base editors(He E-BE4 max and He E-ABEmax)through the fusion of HMG-D protein,which can also improve the transcriptional activation efficiency and base conversion efficiency at multiple endogenous targets,respectively.To evaluate the fidelity of highly enhanced gene-editing tools,we test the off-target efficiency at predicted off-target sites and previously reported off-target sites via HTS,respectively.Results showed that the highly enhanced gene-editing tools also did not detect off-target effects on those sites where without off-target.We also demonstrated that the highly enhanced gene-editing tools can significantly reduce off-target efficiency and improve specificity through delivery of RNP or hp-sg RNA.Most importantly,we found that He E-Sp Cas9 did not significantly increase the number of off-target sites via PEM-seq analysis.Finally,we used AAV8 vector deliver He E-Sp Cas9 /sg RNA to the liver of adult mice,and successfully achieved the high efficiency of Pcsk9 gene editing.Results showed that the gene editing efficiency was increased up to 1.7-fold,and the blood cholesterol of mice was significantly reduced.Collectively,we developed a variety of highly enhanced gene-editing tools by fusing HMG-D protein with Sp Cas9 to enhance DNA binding ability.The highly enhanced gene-editing tool has been successfully implemented in adult mouse liver,and provides a proof-of concept for gene therapy of diseases.