Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection

Du, Ping; Liu, Xiaodan; Yang, Lele; Ren, Naixia; Li, Yingying; Huang, Qilai

doi:10.1089/crispr.2021.0105

Cited by 4 publications

(6 citation statements)

References 36 publications

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“…The mRNA expression levels of EGFP, ABL, CEBPB, or CTCF in the transfected cells before or after cell sorting were determined by qPCR using Taq388 mix ( Du et al, 2022 ) on a QIAGEN Q-Rex machine as previously described ( Ma et al, 2021 ) with primers listed in Supplementary Table S6 . Each pair of PCR primers were tested, and primers with good specificity and amplification efficiency were selected for quantitative PCR analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The genome-editing efficiency was evaluated using the getPCR method ( Li et al, 2019a ) with primers listed in Supplementary Table S6 that had been evaluated for the amplification efficiency and specificity. The qPCR was performed using the Taq 388 mix ( Du et al, 2022 ) on a QIAGEN Q-Rex machine with the program: 5min initial denaturation at 95°C, then 40 cycles of 95°C for 30 s, 67°C for 30 s and 72°C for 15 s with fluorescence acquirement, followed by a final melting curve step increasing from 65°C to 95°C.…”

Section: Methodsmentioning

confidence: 99%

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A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

Yang

Cui

et al. 2022

Front. Bioeng. Biotechnol.

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Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

Yang

Cui

et al. 2022

Front. Bioeng. Biotechnol.

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“…The watching primer used in the indel-sensitive amplicon had three 3 end bases spanning the indel site to distinguish indel alleles from wild-type alleles effectively [24] (Supplementary Figure S1b). The plasmid mixtures mimicking indel frequency of 100%, 50%, 10%, 1%, and 0% were subjected to the get-dPCR assay, using wild-type Taq DNA polymerase (Figure 3a) or Taq388 [38], a highly specific Taq DNA Polymerase (Figure 3b). The results showed that when using the wild-type Taq for get-dPCR, both the wild target sequence and the indel sequences produced almost the same level of FAM fluorescence (Figure 3a).…”

Section: Get-dpcr Distinguishes Indels Clearlymentioning

confidence: 99%

“…The success of this technique is largely defined by the sensitivity of Taq DNA polymerase to primer/template mismatches at the 3 end [37]. Hence, we use an enhanced Taq polymerase generated previously through molecular evolution to improve the ability to discriminate indels from wild sequences [38]. This enhanced Taq DNA polymerase has three amino acid substitutions, S577A, W645R, and I707V, that endow this variant with improved sensitivity to indel-derived primer/template mismatches.…”

Section: Introductionmentioning

confidence: 99%

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations

Li,

Liu,

Huang

2023

IJMS

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Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe’s tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

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“…The mRNA expression levels of given genes in the transfected cells with or without cell sorting enrichment were determined by qPCR using Taq388 mix (Du et al, 2022) on a QIAGEN Q-Rex machine (Qiagen, Germany). The qPCR was performed with the following program: 95 °C for 5 min of initial denaturation, then 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 10 s with fluorescence acquirement, followed by a final melting curve step.…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells

Yang²,

Zuo³

et al. 2022

Front. Mol. Biosci.

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Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

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Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection

Cited by 4 publications

References 36 publications

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells

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