- 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
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.