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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

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Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.

Ping Du¹, Bo Li¹, Xiaodan Liu¹

¹Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, People's Republic of China.

The CRISPR Journal

|January 25, 2022

Summary

This summary is machine-generated.

Researchers developed a highly specific Taq polymerase variant, Taq388, to improve the detection of genome editing. This enhanced DNA polymerase offers superior sensitivity for identifying genetic variations, aiding in CRISPR-Cas9 efficiency evaluation and genotyping.

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Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

Quantitative polymerase chain reaction (qPCR) struggles to differentiate genome editing from wild-type sequences.
A need exists for DNA polymerase variants with enhanced specificity for accurate genome variation detection.

Purpose of the Study:

To develop an improved DNA polymerase variant for sensitive detection of genome editing.
To enhance the specificity of Taq polymerase for applications like CRISPR-Cas9 evaluation and genotyping.

Main Methods:

Semi-rational molecular evolution was employed on full-length Taq polymerase.
Amino acid substitutions and random mutagenesis generated a Taq mutation library.
Screening utilized a qPCR system with insertion and deletion-containing templates.

Main Results:

Identified improved Taq variants, including Taq388 with three amino acid substitutions (S577A, W645R, I707V).
Taq388 demonstrated a significant increase in sensitivity (ΔCt 25-26) to primer/template mismatches caused by insertions and deletions.
The variant showed high potential for allele-specific PCR in single-nucleotide polymorphism detection.

Conclusions:

The Taq388 variant significantly enhances the detection of genome modifications compared to wild-type Taq polymerase.
Taq388 is a valuable tool for evaluating CRISPR-Cas9 editing efficiency and performing single-cell clone genotyping.
This enhanced polymerase holds promise for sensitive single-nucleotide polymorphism detection via allele-specific PCR.