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

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; Gaetan Burgio

doi:10.1186/s13059-019-1776-2

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

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. EudyLin 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, Gaetan Burgio

Research output: Contribution to journal › Article › peer-review

49 Scopus citations

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.

Original language	English (US)
Article number	171
Journal	Genome biology
Volume	20
Issue number	1
DOIs	https://doi.org/10.1186/s13059-019-1776-2
State	Published - Aug 26 2019

Keywords

CRISPR-Cas9
Conditional knockout mouse
Floxed allele
Homology-directed repair
Long single-stranded DNA
Machine learning
Mouse
Oligonucleotide
Reproducibility
Transgenesis

ASJC Scopus subject areas

Ecology, Evolution, Behavior and Systematics
Genetics
Cell Biology

Access to Document

10.1186/s13059-019-1776-2

Cite this

Gurumurthy, C. B., O'Brien, A. R., Quadros, R. M., Adams, J., Alcaide, P., Ayabe, S., Ballard, J., Batra, S. K., Beauchamp, M. C., Becker, K. A., Bernas, G., Brough, D., Carrillo-Salinas, F., Chan, W., Chen, H., Dawson, R., Demambro, V., D'Hont, J., Dibb, K. M., ... Burgio, G. (2019). Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation. Genome biology, 20(1), Article 171. https://doi.org/10.1186/s13059-019-1776-2

Gurumurthy, CB, O'Brien, AR, Quadros, RM, Adams, J, Alcaide, P, Ayabe, S, Ballard, J, Batra, SK, Beauchamp, MC, Becker, KA, Bernas, G, Brough, D, Carrillo-Salinas, F, Chan, W, Chen, H, Dawson, R, Demambro, V, D'Hont, J, Dibb, KM, Eudy, JD, Gan, L, Gao, J, Gonzales, A, Guntur, AR, Guo, H, Harms, DW, Harrington, A, Hentges, KE, Humphreys, N, Imai, S, Ishii, H, Iwama, M, Jonasch, E, Karolak, M, Keavney, B, Khin, NC, Konno, M, Kotani, Y, Kunihiro, Y, Lakshmanan, I, Larochelle, C, Lawrence, CB, Li, L, Lindner, V, Liu, XD, Lopez-Castejon, G, Loudon, A, Lowe, J, Jerome-Majewska, LA, Matsusaka, T, Miura, H, Miyasaka, Y, Morpurgo, B, Motyl, K, Nabeshima, YI, Nakade, K, Nakashiba, T, Nakashima, K, Obata, Y, Ogiwara, S, Ouellet, M, Oxburgh, L, Piltz, S, Pinz, I, Ponnusamy, MP, Ray, D, Redder, RJ, Rosen, CJ, Ross, N, Ruhe, MT, Ryzhova, L, Salvador, AM, Alam, SS, Sedlacek, R, Sharma, K, Smith, C, Staes, K, Starrs, L, Sugiyama, F, Takahashi, S, Tanaka, T, Trafford, AW, Uno, Y, Vanhoutte, L, Vanrockeghem, F, Willis, BJ, Wright, CS, Yamauchi, Y, Yi, X, Yoshimi, K, Zhang, X, Zhang, Y, Ohtsuka, M, Das, S, Garry, DJ, Hochepied, T, Thomas, P, Parker-Thornburg, J, Adamson, AD, Yoshiki, A, Schmouth, JF, Golovko, A, Thompson, WR, Lloyd, KCK, Wood, JA, Cowan, M, Mashimo, T, Mizuno, S, Zhu, H, Kasparek, P, Liaw, L, Miano, JM & Burgio, G 2019, 'Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation', Genome biology, vol. 20, no. 1, 171. https://doi.org/10.1186/s13059-019-1776-2

@article{7084f7ab2c9142e0ad68ce5bafd797af,

title = "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation",

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.",

keywords = "CRISPR-Cas9, Conditional knockout mouse, Floxed allele, Homology-directed repair, Long single-stranded DNA, Machine learning, Mouse, Oligonucleotide, Reproducibility, Transgenesis",

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

note = "Funding Information: All experiments were approved from the respective Institutional Animal Care and Use Committees in the USA and Ethics Committees in Australia, Belgium, the Czech Republic, Japan, Spain, and the UK according to the guidelines or code of practice from the National Institute of Health in the USA, the National Health and Medical Research Council (NHMRC) in Australia, Animals (Scientific Procedures) Act 1986 in the UK or Ministry of Education, Culture, Sports, Science and Technology (MEXT), The Ministry of Health, Labor and Welfare (MHLW) in Japan, the Central Commission for Animal Welfare (CCAW) in the Czech Republic, the Canadian Council on Animal Care (CCAC) in Canada, the National Ethics Code from the Royal Belgian (Flemish) Academy of Medicine in Belgium, and the European Code of Conduct for Research Integrity from All European Academies. Funding Information: All experiments were approved by the respective Institutional Animal Care and Use Committees in the USA and Ethics Committees in Australia, Belgium, the Czech Republic, Japan, Spain, and the UK according to the guidelines or code of practice from the National Institute of Health in the USA, the National Health and Medical Research Council (NHMRC) in Australia, Animals (Scientific Procedures) Act 1986 in the UK, or Ministry of Education, Culture, Sports, Science and Technology (MEXT), The Ministry of Health, Labor and Welfare (MHLW) in Japan, the Central Commission for Animal Welfare (CCAW) in the Czech Republic, the Canadian Council on Animal Care (CCAC) in Canada, the National Ethics Code from the Royal Belgian (Flemish) Academy of Medicine in Belgium, and the European Code of Conduct for Research Integrity from All European Academies. Funding Information: This work was supported by the National Collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network (APN) (to Gaetan Burgio and Paul Thomas), by an Institutional Development Award (PI: Shelley Smith) P20GM103471 (to CBG, RMQ, DWH, JDE, and RR), by NIGMS 1P30GM110768-01 and P30CA036727 (as part of support to University of Nebraska Mouse Genome Engineering and DNA Sequencing Cores), the British Heart Foundation FS12-57, FS12/57/29717, and CH/13/2/30154 and the program grant RG/15/12/31616 (to Kathryn Hentges and Bernard Keavney), the Wellcome Trust grants 107849/Z/ 15/Z, 097820/Z11/B, and 105610/Z/14/Z, the Medical Research Council MR/ N029992/1 (to DB and CBL), the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan, the Canadian Institutes of Health Research MOP#142452 (MCB and LJM). LJM is a member of the Research Centre of the McGill University Health Centre which is supported in part by FQRS. Dr. William Thompson was supported by the Indiana Clinical and Translational Sciences Institute, funded in part by grant #UL1 TR001108 from the National Institute of Health (NIH), National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. KC Kent Lloyd is supported by the NIH (UM1OD023221), and work contributed by staff from the UC Davis Mouse Biology Program (MBP) is supported by a grant from the American College of Laboratory Animal Medicine. The work contributed from Xiande Liu, Chad Smith, Eric Jonasch, Xuesong Zhang, and Jan Parker-Thornburg is supported by the NIH under the award number P30CA16672 (XL, CS, EJ, XZ, JPT) and R50CA211121 (JPT). Joseph Miano is supported by the NIH under the award number HL138987. R Sedlacek was supported by LM2015040 (Czech Centre for Phenogenomics), CZ.1.05/1.1.00/02.0109 (BIOCEV), and CZ.1.05/2.1.00/19.0395 by the Ministry of Education, Youth and Sports (MEYS) and by Academy of Sciences of the Czech Republic (RVO 68378050). David Ray was supported by a Wellcome Trust Investigator (107849/Z/15/Z) and the Medical Research Council (MR/P011853/1 and MR/P023576/) grants. Andrew Loudon was supported by a Wellcome Trust Investigator (107849/Z/15/Z), Biotechnology and Biological Sciences Research Council (BB/N015584/1), Medical Research Council (MR/P023576/1). The work contributed from Gloria Lopez-Castejon is supported by the Wellcome Trust (104192/Z/14/Z) and the Royal Society. Pilar Alcaide was supported by the NIH (HL 123658). The work contributed from Surinder K. Batra is supported by the NIH under the award number P01 CA217798. Publisher Copyright: {\textcopyright} 2019 The Author(s).",

year = "2019",

month = aug,

day = "26",

doi = "10.1186/s13059-019-1776-2",

language = "English (US)",

volume = "20",

journal = "Genome biology",

issn = "1474-7596",

publisher = "BioMed Central",

number = "1",

}

TY - JOUR

T1 - Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles

T2 - A multi-center evaluation

AU - Gurumurthy, Channabasavaiah B.

AU - O'Brien, Aidan R.

AU - Quadros, Rolen M.

AU - Adams, John

AU - Alcaide, Pilar

AU - Ayabe, Shinya

AU - Ballard, Johnathan

AU - Batra, Surinder K.

AU - Beauchamp, Marie Claude

AU - Becker, Kathleen A.

AU - Bernas, Guillaume

AU - Brough, David

AU - Carrillo-Salinas, Francisco

AU - Chan, Wesley

AU - Chen, Hanying

AU - Dawson, Ruby

AU - Demambro, Victoria

AU - D'Hont, Jinke

AU - Dibb, Katharine M.

AU - Eudy, James D.

AU - Gan, Lin

AU - Gao, Jing

AU - Gonzales, Amy

AU - Guntur, Anyonya R.

AU - Guo, Huiping

AU - Harms, Donald W.

AU - Harrington, Anne

AU - Hentges, Kathryn E.

AU - Humphreys, Neil

AU - Imai, Shiho

AU - Ishii, Hideshi

AU - Iwama, Mizuho

AU - Jonasch, Eric

AU - Karolak, Michelle

AU - Keavney, Bernard

AU - Khin, Nay Chi

AU - Konno, Masamitsu

AU - Kotani, Yuko

AU - Kunihiro, Yayoi

AU - Lakshmanan, Imayavaramban

AU - Larochelle, Catherine

AU - Lawrence, Catherine B.

AU - Li, Lin

AU - Lindner, Volkhard

AU - Liu, Xian De

AU - Lopez-Castejon, Gloria

AU - Loudon, Andrew

AU - Lowe, Jenna

AU - Jerome-Majewska, Loydie A.

AU - Matsusaka, Taiji

AU - Miura, Hiromi

AU - Miyasaka, Yoshiki

AU - Morpurgo, Benjamin

AU - Motyl, Katherine

AU - Nabeshima, Yo Ichi

AU - Nakade, Koji

AU - Nakashiba, Toshiaki

AU - Nakashima, Kenichi

AU - Obata, Yuichi

AU - Ogiwara, Sanae

AU - Ouellet, Mariette

AU - Oxburgh, Leif

AU - Piltz, Sandra

AU - Pinz, Ilka

AU - Ponnusamy, Moorthy P.

AU - Ray, David

AU - Redder, Ronald J.

AU - Rosen, Clifford J.

AU - Ross, Nikki

AU - Ruhe, Mark T.

AU - Ryzhova, Larisa

AU - Salvador, Ane M.

AU - Alam, Sabrina Shameen

AU - Sedlacek, Radislav

AU - Sharma, Karan

AU - Smith, Chad

AU - Staes, Katrien

AU - Starrs, Lora

AU - Sugiyama, Fumihiro

AU - Takahashi, Satoru

AU - Tanaka, Tomohiro

AU - Trafford, Andrew W.

AU - Uno, Yoshihiro

AU - Vanhoutte, Leen

AU - Vanrockeghem, Frederique

AU - Willis, Brandon J.

AU - Wright, Christian S.

AU - Yamauchi, Yuko

AU - Yi, Xin

AU - Yoshimi, Kazuto

AU - Zhang, Xuesong

AU - Zhang, Yu

AU - Ohtsuka, Masato

AU - Das, Satyabrata

AU - Garry, Daniel J.

AU - Hochepied, Tino

AU - Thomas, Paul

AU - Parker-Thornburg, Jan

AU - Adamson, Antony D.

AU - Yoshiki, Atsushi

AU - Schmouth, Jean Francois

AU - Golovko, Andrei

AU - Thompson, William R.

AU - Lloyd, K. C.Kent

AU - Wood, Joshua A.

AU - Cowan, Mitra

AU - Mashimo, Tomoji

AU - Mizuno, Seiya

AU - Zhu, Hao

AU - Kasparek, Petr

AU - Liaw, Lucy

AU - Miano, Joseph M.

AU - Burgio, Gaetan

N1 - Funding Information: All experiments were approved from the respective Institutional Animal Care and Use Committees in the USA and Ethics Committees in Australia, Belgium, the Czech Republic, Japan, Spain, and the UK according to the guidelines or code of practice from the National Institute of Health in the USA, the National Health and Medical Research Council (NHMRC) in Australia, Animals (Scientific Procedures) Act 1986 in the UK or Ministry of Education, Culture, Sports, Science and Technology (MEXT), The Ministry of Health, Labor and Welfare (MHLW) in Japan, the Central Commission for Animal Welfare (CCAW) in the Czech Republic, the Canadian Council on Animal Care (CCAC) in Canada, the National Ethics Code from the Royal Belgian (Flemish) Academy of Medicine in Belgium, and the European Code of Conduct for Research Integrity from All European Academies. Funding Information: All experiments were approved by the respective Institutional Animal Care and Use Committees in the USA and Ethics Committees in Australia, Belgium, the Czech Republic, Japan, Spain, and the UK according to the guidelines or code of practice from the National Institute of Health in the USA, the National Health and Medical Research Council (NHMRC) in Australia, Animals (Scientific Procedures) Act 1986 in the UK, or Ministry of Education, Culture, Sports, Science and Technology (MEXT), The Ministry of Health, Labor and Welfare (MHLW) in Japan, the Central Commission for Animal Welfare (CCAW) in the Czech Republic, the Canadian Council on Animal Care (CCAC) in Canada, the National Ethics Code from the Royal Belgian (Flemish) Academy of Medicine in Belgium, and the European Code of Conduct for Research Integrity from All European Academies. Funding Information: This work was supported by the National Collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network (APN) (to Gaetan Burgio and Paul Thomas), by an Institutional Development Award (PI: Shelley Smith) P20GM103471 (to CBG, RMQ, DWH, JDE, and RR), by NIGMS 1P30GM110768-01 and P30CA036727 (as part of support to University of Nebraska Mouse Genome Engineering and DNA Sequencing Cores), the British Heart Foundation FS12-57, FS12/57/29717, and CH/13/2/30154 and the program grant RG/15/12/31616 (to Kathryn Hentges and Bernard Keavney), the Wellcome Trust grants 107849/Z/ 15/Z, 097820/Z11/B, and 105610/Z/14/Z, the Medical Research Council MR/ N029992/1 (to DB and CBL), the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan, the Canadian Institutes of Health Research MOP#142452 (MCB and LJM). LJM is a member of the Research Centre of the McGill University Health Centre which is supported in part by FQRS. Dr. William Thompson was supported by the Indiana Clinical and Translational Sciences Institute, funded in part by grant #UL1 TR001108 from the National Institute of Health (NIH), National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. KC Kent Lloyd is supported by the NIH (UM1OD023221), and work contributed by staff from the UC Davis Mouse Biology Program (MBP) is supported by a grant from the American College of Laboratory Animal Medicine. The work contributed from Xiande Liu, Chad Smith, Eric Jonasch, Xuesong Zhang, and Jan Parker-Thornburg is supported by the NIH under the award number P30CA16672 (XL, CS, EJ, XZ, JPT) and R50CA211121 (JPT). Joseph Miano is supported by the NIH under the award number HL138987. R Sedlacek was supported by LM2015040 (Czech Centre for Phenogenomics), CZ.1.05/1.1.00/02.0109 (BIOCEV), and CZ.1.05/2.1.00/19.0395 by the Ministry of Education, Youth and Sports (MEYS) and by Academy of Sciences of the Czech Republic (RVO 68378050). David Ray was supported by a Wellcome Trust Investigator (107849/Z/15/Z) and the Medical Research Council (MR/P011853/1 and MR/P023576/) grants. Andrew Loudon was supported by a Wellcome Trust Investigator (107849/Z/15/Z), Biotechnology and Biological Sciences Research Council (BB/N015584/1), Medical Research Council (MR/P023576/1). The work contributed from Gloria Lopez-Castejon is supported by the Wellcome Trust (104192/Z/14/Z) and the Royal Society. Pilar Alcaide was supported by the NIH (HL 123658). The work contributed from Surinder K. Batra is supported by the NIH under the award number P01 CA217798. Publisher Copyright: © 2019 The Author(s).

PY - 2019/8/26

Y1 - 2019/8/26

N2 - 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.

AB - 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.

KW - CRISPR-Cas9

KW - Conditional knockout mouse

KW - Floxed allele

KW - Homology-directed repair

KW - Long single-stranded DNA

KW - Machine learning

KW - Mouse

KW - Oligonucleotide

KW - Reproducibility

KW - Transgenesis

UR - http://www.scopus.com/inward/record.url?scp=85071526533&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85071526533&partnerID=8YFLogxK

U2 - 10.1186/s13059-019-1776-2

DO - 10.1186/s13059-019-1776-2

M3 - Article

C2 - 31446895

AN - SCOPUS:85071526533

SN - 1474-7596

VL - 20

JO - Genome biology

JF - Genome biology

IS - 1

M1 - 171

ER -

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

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