Directed Evolution Of Highly Specific Taq And Its Application In Genome Editing Detection

Genomic editing technology is of great significance in gene function research and treatment of genetic diseases.As the third-generation genome editing technology,CRISPR/Cas9 has been widely used in many species since its discovery because of its simplicity,high efficiency,low cost and many other advantages.However,compared with the rapid development of gene editing t echnology,the gene editing frequency detection technology still remains a bottleneck.Existing genetic editing detection techniques include Surveyor assay,Sanger sequencing,PCR-RFLP,HRM,NGS,and the like.Although these methods are widely used,there are many shortcomings.For example,the classic Surveyor assay does not require special equipment,but its quantification is comparatively imprecise,and the operation is cumbersome.The Sanger sequencing method is time consuming,labor intensive,and costly.In order to adapt to the rapid application of gene editing methods,a simple,rapid,economical and accurate gene editing detection method is needed.In our laboratory,we have developed an efficient gene editing detection technology,get PCR.Based on the high specificity of the Taq enzyme in the PCR system,once the indel is generated by the genome editing,the primers and templates are incompletely paired,which cannot be effectively amplified.Selectively quantify the proportion of unedited DNA in quantitative PCR to achieve the detection of genome editing efficiency.The method is easy to operate,accurate,and does not require complicated instruments,and can be completed in ordinary biological laboratories.However,the wild-type Taq enzyme has a certain tolerance to the primer-template mismatch,and in some cases may reduce the accuracy of the detection results.We believed that the main reason for the non-specificity of Taq enzymes was the redundant interactions between Taq and the template-primer complex,which stabilized the complex but made the Taq enzyme insensitive to the mismatch between primertemplate chains.Thus,we attempted to design a library of mutants of Taq DNA polymerase that coupled structure-based rational design and error-prone PCR-mediated random mutation.First,we selected target amino acids for directed mutagenesis that are making direct interaction with the primer/template to reduce excess interactions.Then on this basis,more random mutations were introduced to construct a Taq enzyme mutant library.Furthermore,based on a highly efficient screening system that simultaneously detected activity and specificity of Taq variants,highly specific Taq variants are screened.Among the 40 Taq variants constructed by point mutation method,9 variants completely lost the polymerase activity,and the others showed different degree of improvement in specificity compared with the wild type.On this basis,we further introduced random mutations and selected 39 Taq variants with higher specificity from 1316 clones.After expression and purification in escherichia coli,the activity and specificity of these variants were further systematically evaluated,especially the ability to identify single-base mismatches.Finally,Taq 388 variant was obtained.The variant had high recognition ability and sensitivity to various types of single-base mismatches at the 3' end of primers,and base mismatch of the penultimate position at the 3' end of the primer.It greatly improved the recognition and discrimination ability of getPCR technology for indel generated by genome editing and shows the potential in SNP genotyping applications.