Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology
Authors: Gurumurthy Channabasavaiah , Quadros Rolen , Adams John , Alcaide Pilar , Ayabe Shinya , Ballard Johnathan , Batra Surinder K. , Beauchamp Marie-Claude , Becker Kathleen A , Bernas Guillaume , Brough David , Carrillo-Salinas Francisco , Dawson Ruby , DeMambro Victoria , D’Hont Jinke , Dibb Katharine , Eudy James D. , Gan Lin , Gao Jing , Gonzales Amy , Guntur Anyonya , Guo Huiping , Harms Donald W. , Harrington Anne , Hentges Kathryn E. , Humphreys Neil , Imai Shiho , Ishii Hideshi , Iwama Mizuho , Jonasch Eric , Karolak Michelle , Keavney Bernard , Khin Nay-Chi , Konno Masamitsu , Kotani Yuko , Kunihiro Yayoi , Lakshmanan Imayavaramban , Larochelle Catherine , Lawrence Catherine B. , Li Lin , Lindner Volkhard , Liu Xian-De , Lopez-Castejon Gloria , Loudon Andrew , Lowe Jenna , Jerome-Majeweska Loydie , Matsusaka Taiji , Miura Hiromi , Miyasaka Yoshiki , Morpurgo Benjamin , Motyl Katherine , Nabeshima Yo-ichi , Nakade Koji , Nakashiba Toshiaki , Nakashima Kenichi , Obata Yuichi , Ogiwara Sanae , Ouellet Mariette , Oxburgh Leif , Piltz Sandra , Pinz Ilka , Ponnusamy Moorthy P. , Ray David , Redder Ronald J. , Rosen Clifford J , Ross Nikki , Ruhe Mark T. , Ryzhova Larisa , Salvador Ane M. , Sedlacek Radislav , Sharma Karan , Smith Chad , Staes Katrien , Starrs Lora , Sugiyama Fumihiro , Takahashi Satoru , Tanaka Tomohiro , Trafford Andrew , Uno Yoshihiro , Vanhoutte Leen , Vanrockeghem Frederique , Willis Brandon J. , Wright Christian S. , Yamauchi Yuko , Yi Xin , Yoshimi Kazuto , Zhang Xuesong , Zhang Yu , Ohtsuka Masato , Das Satyabrata , Garry Daniel J. , Hochepied Tino , Thomas Paul , Parker-Thornburg Jan , Adamson Antony D , Yoshiki Atsushi , Schmouth Jean-Francois , Golovko Andrei , Thompson William R. , Lloyd KC. Kent , Wood Joshua A. , Cowan Mitra , Mashimo Tomoji , Mizuno Seiya , Zhu Hao , Kasparek Petr , Liaw Lucy , Miano Joseph M. , Burgio Gaetan 
Published: August 2018

AbstractCRISPR-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.