Site/specificity Study Of CRISPR/Cas9 System And Establishment Of CRISPR/dCas9 Animal Model

Since the genome is recognized as carrier of biological genetic information,scientists have been interested in changing the function of genes,modifying genes to understand functions of biological genes.Scientists committed to the development and improvement of gene editing tools to make it easy and accurate.The rapid improvement of gene editing tools has,in turn,greatly promoted progress in the field of biological sciences.Clustered regularly interspaced short palindromic repeats/CRISPR-associated system(CRISPR/Cas)was originally an acquired immune system of prokaryotes which was used to resist phage and exogenous plasmids.Inspired by acquired immune system of prokaryotes,scientists invented gene edition and modification tool,CRISPR/Cas9.Because of its simplicity and ease of operation,CRISPR/Cas9 has been widely used in the scientific field.It has attacted great interests of doctors and the businessmen that promote CRISPR/Cas9 to clinical treatment of some genetic diseases.At present,CRISPR/Cas9 system has been widely used in the field of gene editing,gene expression regulation,imaging,et al.Scientists will further improve the efficiency of gene editing of CRISPR/Cas9 systems,but also are increasingly concerned about site-specific study of this system.The specificity of the CRISPR/Cas9 system was studied in the previous part of this study.In this study,by an unbiased genome-wide ChIP-seq approach,researchers analyzed binding off-targets of CRISPR/Cas9 in human genome.We found that while Cas9 could bind to various genomic sequences containing PAM and conserved seed sequences in an sgRNA-specific manner,its cleavage off-targets are very limited in comparison with other genome editing enzymes,such as HEases,ZFNs,and TALENs.This might be largely due to additional involvement of the target sequence annealing step in activating the cleavage activities of CRISPR/Cas9 complex on its targets.The study also suggested that binding and cleavage off-targets might be cell type dependent and determined by various complicated factors in addition to primary DNA sequences.This unbiased approach provides a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo applications.In the latter part of this study,SunTag-dCas9 mouse lines were successfully generated,which could express dCas9 in different tissues.This will extend the CRISPR/dCas9 system,which can be used in regulatory gene expression,epigenetic modification,in vivo image,etc,from in vitro studies to in vivo.The SunTag-dCas9 mouse model will be used to establish the animal model to study relationship between gene expression change and diseases,to simulate the occurrence and development of the diseases,to help scientists to understand the pathogenesis of the diseases.The SunTag-dCas9 mouse model will also provide a powerful tool for animal live imaging and promote CRISPR/Cas9 to be used in these fields.