CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR...

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Autores principales: Tanaka, Masayuki, Yokoyama, Keiko, Hayashi, Hideki, Isaki, Sanae, Kitatani, Kanae, Wang, Ting, Kawata, Hisako, Matsuzawa, Hideyuki, Gurumurthy, Channabasavaiah B., Miura, Hiromi, Ohtsuka, Masato
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Method
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9594901/
https://www.ncbi.nlm.nih.gov/pubmed/36284311
http://dx.doi.org/10.1186/s13059-022-02779-8
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author Tanaka, Masayuki
Yokoyama, Keiko
Hayashi, Hideki
Isaki, Sanae
Kitatani, Kanae
Wang, Ting
Kawata, Hisako
Matsuzawa, Hideyuki
Gurumurthy, Channabasavaiah B.
Miura, Hiromi
Ohtsuka, Masato
author_facet Tanaka, Masayuki
Yokoyama, Keiko
Hayashi, Hideki
Isaki, Sanae
Kitatani, Kanae
Wang, Ting
Kawata, Hisako
Matsuzawa, Hideyuki
Gurumurthy, Channabasavaiah B.
Miura, Hiromi
Ohtsuka, Masato
author_sort Tanaka, Masayuki
collection PubMed
description CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02779-8.
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spelling pubmed-95949012022-10-26 CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice Tanaka, Masayuki Yokoyama, Keiko Hayashi, Hideki Isaki, Sanae Kitatani, Kanae Wang, Ting Kawata, Hisako Matsuzawa, Hideyuki Gurumurthy, Channabasavaiah B. Miura, Hiromi Ohtsuka, Masato Genome Biol Method CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02779-8. BioMed Central 2022-10-25 /pmc/articles/PMC9594901/ /pubmed/36284311 http://dx.doi.org/10.1186/s13059-022-02779-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Method
Tanaka, Masayuki
Yokoyama, Keiko
Hayashi, Hideki
Isaki, Sanae
Kitatani, Kanae
Wang, Ting
Kawata, Hisako
Matsuzawa, Hideyuki
Gurumurthy, Channabasavaiah B.
Miura, Hiromi
Ohtsuka, Masato
CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title_full CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title_fullStr CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title_full_unstemmed CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title_short CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice
title_sort crispr-krispr: a method to identify on-target and random insertion of donor dnas and their characterization in knock-in mice
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9594901/
https://www.ncbi.nlm.nih.gov/pubmed/36284311
http://dx.doi.org/10.1186/s13059-022-02779-8
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