An In Vitro Site-directed Mutagenesis Method Based On Combination Of FnCas12a And T5 Exonuclease

Gene editing technology refers to a technology that intentionally inserts,deletes,or mutates single or multiple bases into a target DNA sequence.In recent years,researchers have explored many efficient gene editing tools to make editing genes simple and convenient,such as two endonuclease-based ZENs(Zinc finger nucleases)and TALEN(Transcription activator-like effector nucleases)technologies,and the CRISPR/Cas9 or Cas12 a system mediated editing techniques.Since 2013,gene modification using CRISPR/Cas9 and later CRISPR/Cas variants have become popular.Cas protein is an RNA-mediated endonuclease,and this RNA is a specific sg RNA(Single guide RNA).Under its guidance,the target DNA sequence can be cut and broken.The CRISPR/Cas system is widely used for in vivo and in vitro gene editing because of its high efficiency,ease of operation and accuracy.It can be used to add the required and delete alleles in a single gene at the same time.In this study,we investigated the enzymatic properties of a programmable endonuclease FnCas12a(CRISPR from Prevotella and Francisella,also known as Cpf1)from New Francis.The results show that FnCas12 a fused with CL7 tag can completely cut the target sequence in1-5 minutes and has high cutting efficiency and specificity.At the same time,the results of this paper show that FnCas12 a recognizes 5'-TYN-3' PAM(Protospacer adjacent motif),and cuts randomly on the target and non-target chains at a position about 15-24 nt away from PAM.There are more than 5 cleavage sites on each strand,resulting in non-uniform 5'-sticky ends of 1-8 nt.In order to use FnCas12 a for in vitro gene editing,this paper introduces a T5 exonuclease to repair the ends of DNA fragments produced by FnCas12 a cleavage.And based on this we developed an in vitro site-directed mutagenesis system based on FnCas12 a and T5 exonuclease,called CT5-SDM(FnCas12 a and T5 exonuclease mediated site-directed mutagenesis system).The experimental steps of this method are as follows:(1)Using FnCas12 a RNP to perform single enzyme digestion on the plasmid carrying the gene to be mutated near the site to be mutated,so that the target nucleotide is located on the sticky end resulting from the cleavage.(2)Add an appropriate amount of T5 exonuclease to excise the 5'-sticky ends including the target nucleotides,and form 20-30 nt ss DNA homologous ends.(3)Quickly add single-stranded or double-stranded oligonucleotide fragments containing the required mutant bases(4)Add E.coli clone strain DH5? to the mixture,so that the gaps carried on the plasmid are repaired in E.coli host cells.The study indicated that the mutation efficiency of CT5-SDM can reach 82.7% when double-stranded oligonucleotide fragments are used to mediate mutations,and the mutation efficiency can reach 51.5% when single-stranded oligonucleotide fragments are used to mediate mutations.At the same time,the site-directed mutagenesis method can be used for mutations of vectors larger than 10 kb,with a double-strand mutation efficiency of 43% and a single-strand mutation efficiency of 22%.Further research shows that the principle of CT5-SDM mediated by single-stranded oligonucleotide fragments is different from that of double-stranded oligonucleotide fragments.The former method uses the intracellular homologous recombination system of E.coli.The results of this thesis indicate that ss DNA-mediated homologous recombination in E.coli does not necessarily require the assistance of phage homologous recombination system and high concentration of oligonucleotides,which is another outstanding feature of CT5-SDM.The site-directed mutagenesis technology established in this thesis provides a more effective method for the fields of synthetic biology and protein engineering.At the same time,the combined use of FnCas12 a and T5 exonuclease also provided new ideas for gene cloning.