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

作者：Channabasavaiah B. Gurumurthy, Aidan R. O’Brien, Rolen M. Quadros, John Henry Adams, Pilar Alcaide, Shinya Ayabe, Johnathan L. Ballard, Surinder K. Batra, Marie‐Claude Beauchamp, Kathleen A. Becker, Guillaume Bernas, David Brough, Francisco Javier Carrillo-Salinas, Wesley Chan, Hanying Chen, Ruby Dawson, Victoria DeMambro, Jinke D’Hont, Katharine M. Dibb, James D. Eudy, Lin Gan, Jing Xing Gao, Amy Gonzales, Anyonya R. Guntur, Huiping Guo, Donald W. Harms, Anne N. Harrington, Kathryn E. Hentges, Neil E. Humphreys, Shiho Imai, Hideshi Ishii, Mizuho Iwama, Eric W. Jonasch, Michelle Karolak, Bernard D. 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 López‐Castejón, Andrew S. I. Loudon, Jenna Lowe, Loydie Anne Jerome‐Majewska, Taiji Matsusaka, Hiromi Miura, Yoshiki Miyasaka, Benjamin Morpurgo, Katherine J. Motyl, Yo-ichi Nabeshima, Koji Nakade, Toshiaki Nakashiba, Ken‐ichi Nakashima, Yuichi Obata, Sanae Ogiwara, Mariette Ouellet, Leif H. Oxburgh, Sandra Piltz, Ilka M. Pinz, Moorthy Palanimuthu Ponnusamy, David Ray, Ronald J. Redder, Clifford james Rosen, Nikki Ross, Mark T. Ruhe, Larisa Ryzhova, Ane Miren Salvador, Sabrina Shameen Alam, Radislav Sedláček, Karan Sharma, Chad N. Smith, Katrien Staes, Lora M. Starrs, Fumihiro Sugiyama, Satoru Takahashi, Tomohiro Tanaka, Andrew W. Trafford, Yoshihiro Uno, Leen Vanhoutte, Frederique Vanrockeghem, Brandon Willis, Christian S. Wright, Yuko Yamauchi, Xin Yi, Kazuto Yoshimi, Xuesong Zhang, Yingxin Zhang, Masato Ohtsuka, Satyabrata Das, Daniel J. Garry, Tino Hochepied, Paul Quinton 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 Kašpárek, Lucy Liaw, Joseph M. Miano, Gaétan Burgio · 发表于：Genome biology · 年份：2019 · DOI：10.1186/s13059-019-1776-2 · 被引用次数：111 · 研究领域：CRISPR and Genetic Engineering、Pluripotent Stem Cells Research、Single-cell and spatial transcriptomics

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 mutatio...