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Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.
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- Author(s): Du P;Du P; Li B; Li B; Liu X; Liu X; Yang L; Yang L; Ren N; Ren N; Li Y; Li Y; Huang Q; Huang Q
- Source:
The CRISPR journal [CRISPR J] 2022 Feb; Vol. 5 (1), pp. 131-145. Date of Electronic Publication: 2022 Jan 24.- Publication Type:
Journal Article- Language:
English - Source:
- Additional Information
- Source: Publisher: Mary Ann Liebert, Inc Country of Publication: United States NLM ID: 101738191 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2573-1602 (Electronic) Linking ISSN: 25731599 NLM ISO Abbreviation: CRISPR J Subsets: MEDLINE
- Publication Information: Original Publication: [New Rochelle, NY] : Mary Ann Liebert, Inc., [2018]-
- Subject Terms:
- Abstract: Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.
- Accession Number: EC 2.7.7.- (Taq Polymerase)
- Publication Date: Date Created: 20220125 Date Completed: 20220404 Latest Revision: 20220405
- Publication Date: 20240105
- Accession Number: 10.1089/crispr.2021.0105
- Accession Number: 35076264
- Source:
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