Construction Of Cas9 Temperature Sensitive Mutants Through CRISPR-Cas9 Mediated Site-directed Mutagenesis

The CRISPR-Cas system(clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins)exists in the bacterial and archaeal genomes and is an "adaptive immune defense system" used by these prokaryotic organisms to resist the invasion of foreign DNA such as viruses or plasmids during long-term evolution.In 2012,the CRISPR-Cas system proved to be highly effective in targeting gene modification and could be applied to any tissue,and later proved that the system can effectively modify the mammalian genome specifically,indicating that it has a huge potential application prospects as a gene editing technology.Most of the CRISPR-Cas applications are derived from the CRISPR-Cas9 of the Streptococcus pyogenes,and different CRISPR-Cas systems in other prokaryotes have also been discovered and proved to be effective genetic modification tools.CRISPR-cas9 is an RNA-guided DNA endonuclease that transcribes mature CRISPR RNAs(crRNAs)by directing invading phage or plasmid DNA fragments into CRISPR sequences on the host chromosome,which directs the Cas protein to target Foreign nucleic acid DNA with complementary sequences to guide the degradation of homologous sequences,thereby providing immunity.CRISPR/Cas9 as a new generation of gene editing technology uses a specific RNA-directed endonuclease to target the cleavage of foreign DNA,instead of relying on the protein domain,to complete the gene compared to the traditional ZFN and TALEN technologies.Editing,the operation is more concise and efficient.In order to effectively improve the thermal stability of CRISPR-Cas9,the present study uses the published temperature sensitive mutant prediction site TSpred to analyze Cas9 protein and predict potential sites for mutation,and then uses CRISPR-Cas9-mediated site-directed mutagenesis.The Cas9 gene was digested by the method of transformation,the objective mutation was introduced into the Cas9 gene sequence by PCR,and a Cas9 temperature-sensitive mutant was constructed in combination with the "T5 exonuclease-mediated cloning method"in this laboratory.A Cas9 mutant with high thermal stability was selected,and finally the Cas9 mutation with high thermal stability was selected for expression.The main contents are as follows:1.The sequence of spCas9 protein from Streptococcus pyogenes was found in NCBI.The whole gene sequence was synthesized by Wuhan Jin Kairui and mounted on the pUC57 cloning vector.2.According to the E.coli expression vector pET-23a and Cas9 sequences were designed forward and reverse primers,use PCR amplification of the target gene,the amplified gene fragments recovered,through the laboratory "T5 exonuclease mediated The method of directional cloning of PCR products" was loaded onto the pET-23 a vector.3.Query Cas9's PDB code from the protein database and use the TSpred prediction system to analyze the Cas9 protein for potential thermosensitive mutation sites.4.According to the mutated sites obtained,find appropriate cleavage sites on the Cas9 gene sequence and design primers to synthesize the corresponding sgRNA transcription template.The desired sgRNA was transcribed using the T7 in vitro transcription kit,and the pET23a-Cas9 plasmid was excised together with the purified Cas9 protein.The linearized plasmid recovered was used as a vector for backup,and primers were designed at the cleavage site and the mutant base,respectively.The mutation was introduced into the fragment using PCR.5.The mutated fragment was cloned into the excised vector by T5 cloning.After transformation,the desired Cas9 mutant was picked and sequenced.The Cas9 mutant protein was expressed and purified to detect the effect of different temperature gradients on the original Cas9 protein.Up to now,a total of 70 mutations have been made in 17 loci,among which the selected L279E,L279F and F238G mutant has better cleavage efficiency at 50? than wild-type Cas9,and some of the mutants have a worse cleavage effect at 42? than wild type.For example,Trp located at position 18 of the Cas9 protein sequence has reduced enzyme activity after mutation.Some of the mutants have very low expression levels,such as Leu at position 335 and 380.Due to the large molecular weight of Cas9 protein,the mutation of a single amino acid has little effect on the thermal stability of the entire protein.To find more stable and thermostable mutants,multiple mutants can be constructed by combining several effective mutation sites.Cas9 protein It is very large.Various prediction websites cannot predict it and cannot design multiple point mutations.At present,this part of the work has not yet been carried out.