A Study On FnCpf1-mediated Precise Genome Editing Technology

CRISPR/Cas system(Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein,CRISPR/Cas),as the third-generation gene editing technology,has developed rapidly in recent years.The system uses sg RNA(Single guide RNA,sg RNA)to guide Cas protein to perform targeted cleavage of the target site and activate the non-homologous end joining pathway,resulting in the deletion or insertion of the target site.The system has become an important tool for the gene function identification,which can identify important agronomic traits of crops and promote the genetic improvement of excellent crop traits.However,this technology currently has the bottleneck problem of narrow editing scope and extremely low efficiency of precise gene editing technology.The emergence of single base editing technology(Base editing,BE)has greatly increased the possibility of precise gene editing.However,in single base editing systems,the widely used Cas protein currently mainly uses ‘NGG' as PAM(Protospacer-adjacent motif,PAM)site,which is only suitable for editing DNA sequences enriched in pyrimidine bases.In order to expand the scope of single-base editing,this atricle uses the Fn Cpf1 protein that recognizes the PAM site as ‘TTN' as the effector protein,and constructs the corresponding cytosine base editing system.By using the method of transient transformation of protoplasts,this article tries to take the lead in single-base editing of endogenous maize genes.Through specific amplification of the target site,restriction enzyme digestion screening,sequencing detection and high-throughput sequencing,it is found that the dFnCpf1-CBE system can perform base editing on the target site on the BT2 gene,and there are 9 types of base changes.The highest proportion of reads is the conversion of cytosine nucleotides to adenine nucleotides,and the site editing efficiency is 2.5%.This indicates that the system can recognize ‘TTN' as a PAM site and perform base editing on the target site,but the preference and editing efficiency of its editing sites need to be further explored.The limitation of the editing window is an important factor that affects precise editing.For specific sites,it is still necessary to introduce homologous templates through the HDR approach for precise editing.We use maize,rice and tobacco as research materials to reform the CRISPR/Cas system through the fusion expression of exonuclease,the fusion of Vir D/Vir E protein and the "secondary transformation" method.The aim is to improve the site-specific insertion of foreign fragments more efficiently.After the vector has been transformed into plants,no foreign fragment insertion has been detected by various methods.This may be due to the Cas cleavage activity being affected by the fusion protein,the restriction of transformation methods and detection methods.Subsequently,we need to optimize the experiments for the original fusion expression and test its ability to cleave nucleic acids in vitro,while further improving the transformation and detection methods.To sum up,in terms of base editing,we use the dFnCpf1 protein to expand its editing range.The preliminarily results confirm that the system has a certain editing ability in maize protoplasts.In improving the efficiency of HDR-mediated site-specific insertion,we use a variety of transformation methods in different crops,we also use the improved design of different auxiliary proteins and the design of different exogenous templates.Among them,the site-directed insertion mediated by the fusion expression of Vir protein and nuclease yield preliminary results,but further in-depth testing is still needed.These designs try to provide ideas and support certain data for the subsequent improvement of gene editing technology.