Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

Genome Biology

Masato Ohtsuka¹  Hiromi Miura¹  Catherine Larochelle²  Andrew Loudon³  Hao Zhu⁴  Yu Zhang⁴  Lin Li⁴  Karan Sharma⁵  Shiho Imai⁶  Taiji Matsusaka⁶  Moorthy P. Ponnusamy⁷  Imayavaramban Lakshmanan⁷  Surinder K. Batra⁷  Masamitsu Konno⁸  Tomohiro Tanaka⁹  Jenna Lowe^1,10  Gaetan Burgio^1,10  Nay-Chi Khin^1,10  Lora Starrs^1,10  Nikki Ross^1,10  Jing Gao^1,10  Pilar Alcaide^1,11  Francisco Carrillo-Salinas^1,11  Ane M. Salvador^1,11  Sanae Ogiwara^1,12  Hideshi Ishii^1,13  Loydie A. Jerome-Majewska^1,14  Wesley Chan^1,14  Sabrina Shameen Alam^1,14  Marie-Claude Beauchamp^1,14  Bernard Keavney^1,15  Kathryn E. Hentges^1,16  Catherine B. Lawrence^1,17  David Brough^1,17  Ronald J. Redder^1,18  James D. Eudy^1,18  Seiya Mizuno^1,19  Satoru Takahashi^1,19  Fumihiro Sugiyama^1,19  Yo-ichi Nabeshima^2,20  Petr Kasparek^2,21  Radislav Sedlacek^2,21  Satyabrata Das^2,22  Volkhard Lindner^2,23  Anyonya R. Guntur^2,23  Leif Oxburgh^2,23  Anne Harrington^2,23  Clifford J. Rosen^2,23  Victoria DeMambro^2,23  Michelle Karolak^2,23  Ilka Pinz^2,23  Larisa Ryzhova^2,23  Kathleen A. Becker^2,23  Katherine Motyl^2,23  Lucy Liaw^2,23  Gloria Lopez-Castejon^2,24  Mitra Cowan^2,25  Brandon J. Willis^2,26  Joshua A. Wood^2,26  Mark T. Ruhe^2,26  K. C. Kent Lloyd^2,26  Channabasavaiah B. Gurumurthy^2,27  Rolen M. Quadros^2,27  Donald W. Harms^2,27  David Ray^2,28  Daniel J. Garry^2,29  Koji Nakade^3,30  Toshiaki Nakashiba^3,30  Atsushi Yoshiki^3,30  Kenichi Nakashima^3,30  Shinya Ayabe^3,30  Yuichi Obata^3,30  Mizuho Iwama^3,30  William R. Thompson^3,31  Xin Yi^3,31  Christian S. Wright^3,31  Hanying Chen^3,32  Ruby Dawson^3,33  Paul Thomas^3,33  Sandra Piltz^3,33  Andrei Golovko^3,34  John Adams^3,34  Johnathan Ballard^3,34  Huiping Guo^3,34  Amy Gonzales^3,34  Benjamin Morpurgo^3,34  Yoshiki Miyasaka^3,35  Yoshihiro Uno^3,35  Yuko Kotani^3,35  Yayoi Kunihiro^3,35  Kazuto Yoshimi^3,35  Yuko Yamauchi^3,35  Tomoji Mashimo^3,35  Xuesong Zhang^3,36  Eric Jonasch^3,36  Xian-De Liu^3,36  Chad Smith^3,36  Jan Parker-Thornburg^3,36  Aidan R. O’Brien^3,37  Jean-Francois Schmouth^3,38  Mariette Ouellet^3,38  Guillaume Bernas^3,38  Leen Vanhoutte^3,39  Frederique Vanrockeghem^3,39  Katrien Staes^3,39  Tino Hochepied^3,39  Jinke D’Hont^3,39  Antony D. Adamson^4,40  Neil Humphreys^4,40  Andrew W. Trafford^4,41  Katharine M. Dibb^4,41  Lin Gan^4,42  Joseph M. Miano^4,42 

[1]Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University

[2]Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM)

[3]Centre for Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester

[4]Children’s Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center

[5]College of Osteopathic Medicine, Marian University

[6]Department of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University

[7]Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center

[8]Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine

[9]Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences

[10]Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University

[11]Department of Immunology, Tufts University School of Medicine

[12]Department of Laboratory Animal Science, Support Center for Medical Research and Education, Tokai University

[13]Department of Medical Data Science, Osaka University Graduate School of Medicine

[14]Departments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC)

[15]Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University of Manchester and Manchester Heart Centre, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre

[16]Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester

[17]Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester

[18]High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center

[19]Laboratory Animal Resource Center, University of Tsukuba

[20]Laboratory of Molecular Life Science, Foundation for Biomedical Research and Innovation

[21]Laboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences

[22]Lillehei Heart Institute Regenerative Medicine and Sciences Program, University of Minnesota

[23]Maine Medical Center Research Institute (MMCRI)

[24]Manchester Collaborative Centre for Inflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester

[25]McGill Integrated Core for Animal Modeling (MICAM)

[26]Mouse Biology Program, University of California

[27]Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center

[28]Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford

[29]Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota

[30]RIKEN BioResource Research Center

[31]School of Health and Human Sciences, Department of Physical Therapy, Indiana University

[32]School of Medicine, Indiana University

[33]South Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide

[34]Texas A&M Institute for Genomic Medicine (TIGM), Texas A&M University

[35]The Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine

[36]The University of Texas MD Anderson Cancer Center

[37]Transformational Bioinformatics, Health and Biosecurity Business Unit, CSIRO

[38]Transgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM)

[39]Transgenic Mouse Core Facility, VIB Center for Inflammation Research

[40]Transgenic Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester

[41]Unit of Cardiac Physiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester

[42]University of Rochester Medical Center

关键词: CRISPR-Cas9;    Mouse;    Transgenesis;    Homology-directed repair;    Conditional knockout mouse;    Floxed allele;   

DOI  :  10.1186/s13059-019-1776-2

来源: DOAJ

【 摘 要 】

Abstract Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

【 授权许可】

Unknown