| The autoimmune defense mechanism found in bacteria and archaea has been artificially modified and widely used in scientific research.It was officially named the CRISPR/Cas system in 2002.Only a single-stranded RNA can guide the effector to a specific target and make effective cuts.Class 2 effectors of type V have attracted much attention due to their characteristics such as cutting away from the PAM sequence and forming sticky ends.The AsCas12a protein in this type of effector has been studied extensively because of its good editing activity on mammalian cells.Little effort has been made to expand its PAM recognition specificity to broaden its target range.LbCas12a from Lachnospiraceae bacterium ND2006 shows good editing activity in plant genome editing,but there are few studies on expanding the PAM recognition ability of this protein.With the deepening of plant genome research,it is necessary to expand the scope of targeted editing of the protein.Therefore,this article conducts research from the following three aspects.First of all,the prerequisite for conducting research is to determine the mutation range of the protein.The crystal structure of the protein has been resolved.Existing research work has shown that the closest amino acid around the PAM double-strand structure is the key amino acid that affects the recognition ability of PAM.Therefore,after downloading the crystal structure of LbCas12a protein in the protein database PBD,the key sites were found through Pymol software.There are 10 key sites,namely T149,T152,D156,Q529,G532,K538,D541,Y542,K591,K595.Secondly,in order to understand the influence of amino acid residues obtained from spatial structure information on the PAM recognition ability of LbCas12a protein,a negative screening system was constructed and used to perform alanine scanning on 10 alanine mutants,and 10 sites were confirmed one by one.Affect the degree of PAM recognition ability,so as to determine the core key sites.The results show that 4 of the 10 loci have a significant impact on the recognition ability of the protein wild-type PAM.According to the data analysis and summary,the degree of impact is K595,T149,K538/D156,and the remaining 6 loci.The introduction of point alanine mutations had no effect on the recognition ability of wild-type PAM.Finally,in order to more comprehensively investigate the impact of key sites on PAM recognition specificity,10 sites were mutated to arginine,6 N random PAM libraries were constructed,and then a negative screening system combined with high-throughput sequencing and biological Informatics processing confirms the preference of mutant and wild-type protein for PAM.The analysis results show that the wild-type protein showed a specific preference for TYTV in this experiment.The introduction of ten-site single amino acid mutations has four different effects on the specific recognition of PAM.One is that the introduction of mutations has four different effects on the protein PAM.There is no effect on recognition preference.These mutations are T149R,T152R,D156R,Q529R,D541R,Y542R.Second,the introduction of mutations strengthens the preference for T at position d(-3*).The mutations that have this effect are G532R,K538R,The third is the introduction of mutations to expand the recognition of d(-2*)to W(A/T),that is,the K595R mutation,and the fourth is the introduction of mutations to expand the recognition of d(-2*)to W(A/T).T),while also enhancing the recognition preference of d(-3*)to T,that is,the K591R mutation.Randomly select each type of site,namely T152,G532,K591,K595 for random combination mutations,and get 11 combined mutants.The negative screening system confirms the PAM recognition preference of combined mutants,and obtains K591R/K595R that can recognize non-standard PAM.Mutants,their relaxed PAM are TYWV.In order to verify this result,a positive screening system was constructed to verify its relaxation at position d(-2*),and the results confirmed the ability of the mutant to recognize A at position d(-2*). |
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