Preliminary Research On A Cytosine Base Editor Based On CRISPR/Cas9

CRISPR/ Cas9 technology introduces double-stranded DNA breaks(DSBs)at a target locus as the first step to make gene corrections,which easily results in undesired mutations.Thus,The researchers develop new methods-Base editing,Base editors directly convert one base or base pair into another,enabling the efficient installation of point mutations in cells Furthermore,CRISPR/Cas9-derived base editing also improves efficiency in repairing point mutations in the genome.In this project,the HNH domain and Ruvc-like domain of CRISPR/ Cas9 protein were replaced by human cytosine deaminase(AID),and a new base editor was redesigned to detect the base editing window and editing efficiency of this new base editor.First,an in vitro functional experiment was conducted on the base editor.Eight dCas9 recombinant plasmids were obtained by replacing the HNH domain and the RuvC-like domain in sp Cas9 with AID protein,and then connecting UGI sequence at the N terminal of the protein to obtain dcas9-aid protein.Meanwhile,specific sgRNA was designed in vitro,and 100 bp sgRNA was obtained through in vitro transcriptional purification and other steps.The substrate DNA sequence was designed according to the sequence of sgRNA,and a large number of DNA fragments were obtained by PCR amplification.Using prokaryotic expression system in vitro protein purification base editor,the recombinant proteins for GST affinity chromatography?Ni affinity chromatography?Heparin agarose gel chromatography column(Heparin)and anion exchange chromatography column(S),the existence of ultraviolet absorption sds-page gel electrophoresis,the samples will be purified to obtain the fusion protein with advance design good sgRNA and mixed substrate DNA,the activity of in vitro testing base editor.The results showed that the protein was not expressed correctly and the in vitro functional experiment failed.Then pCMV-BE4 max for intracellular expression vector for template modification,received twelve different kinds of restructuring the carrier,and determined by the literature FMN1 gene loci for the purpose,based on the design purpose gene sequences sgRNA and connects the pU6 carrier,to construct the eukaryotic expression vector into 293 T cells,the detection base editor in the body function,through WB and genome sequencing analysis,found the bases editor protein expression in cells in normal and the cells genome PCR amplification,after extracting the purpose gene sequencing.The ability of the protein to base edit was not detected.This experiment confirmed that domain replacement of CRISPR/ Cas9 protein was not feasible,possibly because the protein function was abnormal due to structural damage of the protein,and it was unable to recognize specific sites and conduct base editing.