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

SUGIYAMA Fumihiro, TAKAHASHI Satoru, MIZUNO Seiya, Gurumurthy Channabasavaiah B., O’Brien Aidan R., Quadros Rolen M., Adams John, Alcaide Pilar, Ayabe Shinya, Ballard Johnathan, Batra Surinder K., Beauchamp Marie-Claude, Becker Kathleen A., Bernas Guillaume, Brough David, Carrillo-Salinas Francisco, Chan Wesley, Chen Hanying, Dawson Ruby, DeMambro Victoria, D’Hont Jinke, Dibb Katharine M., Eudy James D., Gan Lin, Gao Jing, Gonzales Amy, Guntur Anyonya R., 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-Majewska Loydie A., 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., Alam Sabrina Shameen, Sedlacek Radislav, Sharma Karan, Smith Chad, Staes Katrien, Starrs Lora, Tanaka Tomohiro, Trafford Andrew W., 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 K. C. Kent, Wood Joshua A., Cowan Mitra, Mashimo Tomoji, Zhu Hao, Kasparek Petr, Liaw Lucy, Miano Joseph M., Burgio Gaetan

doi:10.1186/s13059-019-1776-2

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>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).</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>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.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in <jats:italic>cis</jats:italic>, 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.</jats:p> </jats:sec>

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

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

この論文をさがす

説明

収録刊行物

被引用文献 (11)*注記

参考文献 (39)*注記

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