Significantly Improved HDR Gene Knock-in Efficiencies By Exploring The Mechanism Of DsDNA Repair After CRISPR-Cas9 Cleavage

Objective:It has been an important project to edit patient-specific induced pluripotent stem cell(iPSC)genomes for clinical gene and cell therapy.CRISPR-Cas9 is a robust tool for gene knockout(KO).However,detailed investigation for homology-directed repair(HDR)-mediated gene knock-in(K1)was still lacking.Therefore,we were looking forward to exploring the detailed characteristics of DNA double-strand break(DSB)repair profiles mediated by CRISPR-Cas9 genome editing,and improving genome editing,particularly HDR precise gene KI efficiencies.Besides,the KI reporter gene expression level was to be improved without sacrificing the high KI efficiency through modified HDR templates.Methods:Firstly,we designed a series of sgRNAs targeting several genes at different loci.These sgRNAs were in context diversities:with NHEJ(Non-homologous end joining)-or MMEJ(Microhomology-mediated end joining)-prone.Commercially available crRNAs(CRISPR RNAs)were bought and annealed with tracrRNAs(trans-activating crispr RNAs)to form gRNAs.Cas9 protein and gRNAs can combine to form RNP with electroporation stability after incubation at room temperature.Well-conditioned iPSCs or other cell lines were electroporated with RNP.Then AAV6 HDR donors or small molecular compounds were added into the culture medium according to requirements.Edited cells were harvested at different time points,followed by genomic DNA(gDNA)extraction,PCR amplification,and Illumina high-throughput sequencing.Comprehensive visualization results were generated after data processing and analysis,including total editing efficiencies,each repair outcome,and their frequencies.Besides,we also utilized the plasmid delivery system and our previously developed double-cut HDR donors to KI a fluorescence protein reporter gene as well as determined the positive percentage and expression intensity of the fluorescence protein.Results:We compared the gene-editing efficiencies at multiple gene loci in several cell types and demonstrated that the editing efficiencies of the same cell type at different gene loci,or the same gene locus in different cell types,were all distinctive.Investigating the TPM(RNA-seq transcripts per million)values of these targeted genes,we demonstrated that the editing efficiencies of transcriptionally active genes(with high TPM values)were significantly higher than transcriptionally silent genes,in which the TPM values were closed to zero.We collected and analyzed the T50 of each DNA repair pattern after CRISPR-Cas9 RNP editing,which suggested the dynamics or speed of each repair pathway.We demonstrated that the T50 values of NHEJ+1 were the lowest.The T50 values of MMEJ were higher,while HDR was placed in between.Besides,MMEJ-mediated shorter-length deletion occurs faster than medium-length deletion,while the T50 values of MMEJmediated long-length(>10 bp)deletions were the highest.It is the first time that we explored and revealed the dynamics and characteristics of CRISPR-Cas9 RNP editing outcomes mediated by different canonical DNA repair pathways.The relative frequencies of MMEJ significantly decreased when the AAV6 HDR donor presented.After the addition of small molecular NHEJ inhibitors,the proportion of NHEJ dropped sharply,while the HDR efficiencies were significantly improved.We for the first time demonstrated the competition of these three canonical DNA repair pathways and utilized robust NHEJ inhibitors to improve the HDR mediated precise gene KI efficiencies in iPSCs and other clinically relevant cell types.It has been demonstrated that many intron regions contain cis-regulatory elements such as proximal or distal enhancer-like signatures.The reporter gene expression levels were improved with our novel intron retention strategies or artificial intron-containing HDR donors.Conclusions:1.In all the tested cell types,transcriptionally active genes are more accessible for gene editing.2.CRISPR-Cas9 editing efficiencies and DNA repair outcomes are largely determined by the sgRNA context itself.We observed the strong bias of duplications of the-4 nucleotide before protospacer adjacent motif(PAM)when the-4 position is A or T.3.After CRISPR-Cas9 mediated DSB,three main DNA repair pathways show distinct dynamics:occurrence speed NHEJ>HDR>MMEJ.4.The addition of small molecules such as HD AC inhibitors and robust NHEJ inhibitors can improve genome editing efficiencies,especially HDR efficiencies.M3814 increases HDR editing efficiency mainly by blocking NHEJ events.5.The presence of artificial introns,particularly an intron that carries an enhancer element,significantly increased the reporter expression levels in iPSCs.