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1. Response to correspondence on 'Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation'

Author

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 Dibb, James D. Eudy, Lin Gan, Jing Gao, Amy Gonzales, Anyonya 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 Jerome-Majeweska, 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 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, and Gaetan Burgio

Subjects
Biology (General), QH301-705.5, Genetics, QH426-470
Published
2021
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2. Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

Author

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, and Gaetan Burgio

Subjects
CRISPR-Cas9, Mouse, Transgenesis, Homology-directed repair, Conditional knockout mouse, Floxed allele, Biology (General), QH301-705.5, Genetics, QH426-470
Abstract

Abstract 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 mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Published
2019
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3. Response to correspondence on 'Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation'

Author

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

Subjects
Gene Editing, lcsh:QH426-470, Biology and Life Sciences, Reproducibility of Results, Computational biology, Biology, Human genetics, lcsh:Genetics, Mice, lcsh:Biology (General), CRISPR-Associated Protein 9, Correspondence, CRISPR, Animals, Center (algebra and category theory), Allele, CRISPR-Cas Systems, lcsh:QH301-705.5, Alleles
Published
2021

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