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 & 93 others 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

Research output: Contribution to journalArticle

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.

LanguageEnglish
Article number171
JournalGenome Biology
Volume20
Issue number1
DOIs
Publication statusPublished - 26 Aug 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

Cite this

Gurumurthy, C. B., O'Brien, A. R., Quadros, R. M., Adams, J., Alcaide, P., Ayabe, S., ... Burgio, G. (2019). Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation. Genome Biology, 20(1), [171]. https://doi.org/10.1186/s13059-019-1776-2
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 ; Sugiyama, Fumihiro ; Takahashi, Satoru ; 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 ; Mizuno, Seiya ; Zhu, Hao ; Kasparek, Petr ; Liaw, Lucy ; Miano, Joseph M. ; Burgio, Gaetan. / Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles : A multi-center evaluation. In: Genome Biology. 2019 ; Vol. 20, No. 1.
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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.",
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month = "8",
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doi = "10.1186/s13059-019-1776-2",
language = "English",
volume = "20",
journal = "Genome biology",
issn = "1465-6906",
publisher = "BioMed Central",
number = "1",

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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

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles : A multi-center evaluation. / 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; Sugiyama, Fumihiro; Takahashi, Satoru; 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; Mizuno, Seiya; Zhu, Hao; Kasparek, Petr; Liaw, Lucy; Miano, Joseph M.; Burgio, Gaetan.

In: Genome Biology, Vol. 20, No. 1, 171, 26.08.2019.

Research output: Contribution to journalArticle

TY - JOUR

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

T2 - Genome biology

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

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

U2 - 10.1186/s13059-019-1776-2

DO - 10.1186/s13059-019-1776-2

M3 - Article

VL - 20

JO - Genome biology

JF - Genome biology

SN - 1465-6906

IS - 1

M1 - 171

ER -