Rapid And Efficient Site-directed Mutagenesis By A PCR-free In Vitro CRISPR/Cas9-mediated Mutagenic System

Site-directed mutagenesis is a technique for constructing user-defined mutants of target gene,which has been widely used for directed evolution and investigation of correlation between the sequence and function,thus generating protein with better properties to meet the requirements of biomedical,biotechnology and enzyme engineering industry.While some technologies to establish mutant library has been reported up to now,there still existed some technical limitations.Recently,the CRISPR/Cas9 system,a powerful specific RNA-guided nuclease,has been widely used in genome editing with high efficiency in vivo,but the application in vitro has been rarely reported.In the quest to overcome the inherent limitations encountered in the traditional PCR-based approaches,we present here an in vitro CRISPR/Cas9-mediated mutagenic(ICM)system as a paradigm shift in site-directed mutagenesis.Firstly,it involves a digestion of target plasmid by utilizing the complex of Cas9 protein and specific single guide RNA(sgRNA),followed by pretreatment with T5 exonuclease to remove base in 5' to 3' direction,thus generating the linearized plasmid with 15nt sticky end homologous with mutagenic primer.Secondly,a pair/set of primers containing the desired mutation is annealed to serve as inserted fragment,and then ligated into linearized plasmid by co-transformation into host E.coli cells.The main contents of our research are as follows:1.Expression and purification of CRISPR-Cas9 proteinGene sequence encoding CRISPR-Cas9 protein was synthesized and cloned into pET23a plasmid for constructing expression plasmid pET23a-Cas9,which was then transformed into host cell.Then single strain was inoculated,and Cas9 protein was purified and obtained by Ni-nitrilotriacetic acid(Ni-NTA)agarose,finally stored for subsequent experiments;2.Site-directed mutagenesis by ICM systemTo evaluate the performance of CRISPR/Cas9-mediated mutagenic(ICM)system,we first tested its ability for single site-directed mutagenesis.Single-site mutagenesis of green fluorescent protein(GFP)gene was successfully performed by ICM system with more than 95%efficiency.We next attempted to introduce double site-directed mutagenesis of GFP gene.Then we successfully constructed three mutants of the HHT2 gene at adjacent sites with the same plasmid backbone.Besides,we achieved mutagenesis of a 9.5 kb large plasmid(pPIC9K-PAT4)with approximately 80%efficiency,which reveals that our method can also be used as a powerful tool to manipulate large plasmids with high efficiency,thus overcoming the limitation of conventional PCR approach in amplifying over-size products;3.Site-saturation mutagenesis by ICM systemIn addition to the site-directed mutagenesis,the ICM system mediated site saturation mutagenesis(SM)was also attempted for SM library construction.Firstly,NNK-based primers were utilized to achieve single-site SM of GFP by ICM-based and PCR-based approachs in parallel.The results showed that ICM-based library presentated higher genetic diversity than PCR-based library;then NNN-based primers were utilized to perform double-site SM.Likewise,ICM-based library was also superior to PCR-based library in terms of bias of nucleotide and amino acid;finally,rationally chosen reduced amino acid alphabets were successfully introduced by ICM system to construct a comprehensive combinatorial library on four sites of a limonene epoxide hydrolase(LEH),all four amino acids(parental amino acid,V,F and Y)can be observed at each position and residue distribution is in good agreement with the designed values;4.The application of ICM system in investigating correlation between sequence and function of proteinTo further expand the utility of ICM system,GGN(SUMO protease cleavage site,N for 19 kinds of amino acids except serine)of LY338-SUMO target plasmid was selected as mutational site to construct a series of mutations at specific position and find the relationship between sequences and function.All 19 desired mutants were obtained and displayed on surface Saccharomyces cerevisiae cell,and the relationship between protease digestion efficiency and amino-acid residues followed by the cleavage site was verified.ICM system has successfully and efficiently achieved the single and multi-site mutagenesis,and even saturated mutagenesis.Due to wider applicability and simplification than traditional method,it can be expected to facilitate research in synthetic biology.