著者

杉山 文博 高橋 智 水野 聖哉 Channabasavaiah B. Gurumurthy Aidan R. O'Brien Rolen M. Quadros John Adams Pilar Alcaide Shinya Ayabe Johnathan Ballard Surinder K. Batra Marie-Claude Beauchamp Kathleen A. Becker Guillaume Bernas David Brough Francisco Carrillo-Salinas Wesley Chan Hanying Chen Ruby Dawson Victoria DeMambro Jinke D'Hont Katharine M. Dibb James D. Eudy Lin Gan Jing Gao Amy Gonzales Anyonya R. Guntur Huiping Guo Donald W. Harms Anne Harrington Kathryn E. Hentges Neil Humphreys Shiho Imai Hideshi Ishii Mizuho Iwama Eric Jonasch Michelle Karolak Bernard Keavney Nay-Chi Khin Masamitsu Konno Yuko Kotani Yayoi Kunihiro Imayavaramban Lakshmanan Catherine Larochelle Catherine B. Lawrence Lin Li Volkhard Lindner Xian-De Liu Gloria Lopez-Castejon Andrew Loudon Jenna Lowe Loydie A. Jerome-Majewska Taiji Matsusaka Hiromi Miura Yoshiki Miyasaka Benjamin Morpurgo Katherine Motyl Yo-ichi Nabeshima Koji Nakade Toshiaki Nakashiba Kenichi Nakashima Yuichi Obata Sanae Ogiwara Mariette Ouellet Leif Oxburgh Sandra Piltz Ilka Pinz Moorthy P. Ponnusamy David Ray Ronald J. Redder Clifford J. Rosen Nikki Ross Mark T. Ruhe Larisa Ryzhova Ane M. Salvador Sabrina Shameen Alam Radislav Sedlacek Karan Sharma Chad Smith Katrien Staes Lora Starrs Fumihiro SUGIYAMA Satoru TAKAHASHI Tomohiro Tanaka Andrew W. Trafford Yoshihiro Uno Leen Vanhoutte Frederique Vanrockeghem Brandon J. Willis Christian S. Wright Yuko Yamauchi Xin Yi Kazuto Yoshimi Xuesong Zhang Yu Zhang Masato Ohtsuka Satyabrata Das Daniel J. Garry Tino Hochepied Paul Thomas Jan Parker-Thornburg Antony D. Adamson Atsushi Yoshiki Jean-Francois Schmouth Andrei Golovko William R. Thompson K. C. Kent Lloyd Joshua A. Wood Mitra Cowan Tomoji Mashimo Seiya MIZUNO Hao Zhu Petr Kasparek Lucy Liaw Joseph M. Miano Gaetan Burgio

出版者

BMC

雑誌

Genome Biology (ISSN:1474760X)

巻号頁・発行日

vol.20, no.1, 2019-08

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BackgroundCRISPR-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).ResultsWe 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.ConclusionWe 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.