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Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl(2) concentratio...

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Autores principales: Sakurai, Takayuki, Kamiyoshi, Akiko, Takei, Norio, Watanabe, Satoshi, Sato, Masahiro, Shindo, Takayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617447/
https://www.ncbi.nlm.nih.gov/pubmed/31289302
http://dx.doi.org/10.1038/s41598-019-46357-8
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author Sakurai, Takayuki
Kamiyoshi, Akiko
Takei, Norio
Watanabe, Satoshi
Sato, Masahiro
Shindo, Takayuki
author_facet Sakurai, Takayuki
Kamiyoshi, Akiko
Takei, Norio
Watanabe, Satoshi
Sato, Masahiro
Shindo, Takayuki
author_sort Sakurai, Takayuki
collection PubMed
description We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl(2) concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene’s proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl(2) concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl(2) concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).
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spelling pubmed-66174472019-07-18 Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase Sakurai, Takayuki Kamiyoshi, Akiko Takei, Norio Watanabe, Satoshi Sato, Masahiro Shindo, Takayuki Sci Rep Article We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl(2) concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene’s proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl(2) concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl(2) concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR). Nature Publishing Group UK 2019-07-09 /pmc/articles/PMC6617447/ /pubmed/31289302 http://dx.doi.org/10.1038/s41598-019-46357-8 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Sakurai, Takayuki
Kamiyoshi, Akiko
Takei, Norio
Watanabe, Satoshi
Sato, Masahiro
Shindo, Takayuki
Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title_full Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title_fullStr Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title_full_unstemmed Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title_short Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase
title_sort bindel-pcr: a novel and convenient method for identifying crispr/cas9-induced biallelic mutants through modified pcr using thermus aquaticus dna polymerase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617447/
https://www.ncbi.nlm.nih.gov/pubmed/31289302
http://dx.doi.org/10.1038/s41598-019-46357-8
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