TY - JOUR T1 - Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology JF - bioRxiv DO - 10.1101/393231 SP - 393231 AU - Channabasavaiah Gurumurthy AU - Rolen Quadros AU - John Adams, Jr AU - Pilar Alcaide AU - Shinya Ayabe AU - Johnathan Ballard AU - Surinder K. Batra AU - Marie-Claude Beauchamp AU - Kathleen A Becker AU - Guillaume Bernas AU - David Brough AU - Francisco Carrillo-Salinas AU - Ruby Dawson AU - Victoria DeMambro AU - Jinke D’Hont AU - Katharine Dibb AU - James D. Eudy AU - Lin Gan AU - Jing Gao AU - Amy Gonzales AU - Anyonya Guntur AU - Huiping Guo AU - Donald W. Harms AU - Anne Harrington AU - Kathryn E. Hentges AU - Neil Humphreys AU - Shiho Imai AU - Hideshi Ishii AU - Mizuho Iwama AU - Eric Jonasch AU - Michelle Karolak AU - Bernard Keavney AU - Nay-Chi Khin AU - Masamitsu Konno AU - Yuko Kotani AU - Yayoi Kunihiro AU - Imayavaramban Lakshmanan AU - Catherine Larochelle AU - Catherine B. Lawrence AU - Lin Li AU - Volkhard Lindner AU - Xian-De Liu AU - Gloria Lopez-Castejon AU - Andrew Loudon AU - Jenna Lowe AU - Loydie Jerome-Majeweska AU - Taiji Matsusaka AU - Hiromi Miura AU - Yoshiki Miyasaka AU - Benjamin Morpurgo AU - Katherine Motyl AU - Yo-ichi Nabeshima AU - Koji Nakade AU - Toshiaki Nakashiba AU - Kenichi Nakashima AU - Yuichi Obata AU - Sanae Ogiwara AU - Mariette Ouellet AU - Leif Oxburgh AU - Sandra Piltz AU - Ilka Pinz AU - Moorthy P. Ponnusamy AU - David Ray AU - Ronald J. Redder AU - Clifford J Rosen AU - Nikki Ross AU - Mark T. Ruhe AU - Larisa Ryzhova AU - Ane M. Salvador AU - Radislav Sedlacek AU - Karan Sharma AU - Chad Smith AU - Katrien Staes AU - Lora Starrs AU - Fumihiro Sugiyama AU - Satoru Takahashi AU - Tomohiro Tanaka AU - Andrew Trafford AU - Yoshihiro Uno AU - Leen Vanhoutte AU - Frederique Vanrockeghem AU - Brandon J. Willis AU - Christian S. Wright AU - Yuko Yamauchi AU - Xin Yi AU - Kazuto Yoshimi AU - Xuesong Zhang AU - Yu Zhang AU - Masato Ohtsuka AU - Satyabrata Das AU - Daniel J. Garry AU - Tino Hochepied AU - Paul Thomas AU - Jan Parker-Thornburg AU - Antony D Adamson AU - Atsushi Yoshiki AU - Jean-Francois Schmouth AU - Andrei Golovko AU - William R. Thompson AU - KC. Kent Lloyd AU - Joshua A. Wood AU - Mitra Cowan AU - Tomoji Mashimo AU - Seiya Mizuno AU - Hao Zhu AU - Petr Kasparek AU - Lucy Liaw AU - Joseph M. Miano AU - Gaetan Burgio Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/08/30/393231.abstract N2 - CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes. ER -