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

C.B. Gurumurthy, A.R. O'Brien, R.M. Quadros, J. Adams, P. Alcaide, S. Ayabe, J. Ballard, S.K. Batra, M.-C. Beauchamp, K.A. Becker, G. Bernas, D. Brough, F. Carrillo-Salinas, W. Chan, H. Chen, R. Dawson, V. Demambro, J. D'Hont, K.M. Dibb, J.D. EudyL. Gan, J. Gao, A. Gonzales, A.R. Guntur, H. Guo, D.W. Harms, A. Harrington, K.E. Hentges, N. Humphreys, S. Imai, H. Ishii, M. Iwama, E. Jonasch, M. Karolak, B. Keavney, N.-C. Khin, M. Konno, Y. Kotani, Y. Kunihiro, I. Lakshmanan, C. Larochelle, C.B. Lawrence, L. Li, V. Lindner, X.-D. Liu, G. Lopez-Castejon, A. Loudon, J. Lowe, L.A. Jerome-Majewska, T. Matsusaka, H. Miura, Y. Miyasaka, B. Morpurgo, K. Motyl, Y.-I. Nabeshima, K. Nakade, T. Nakashiba, K. Nakashima, Y. Obata, S. Ogiwara, M. Ouellet, L. Oxburgh, S. Piltz, I. Pinz, M.P. Ponnusamy, D. Ray, R.J. Redder, C.J. Rosen, N. Ross, M.T. Ruhe, L. Ryzhova, A.M. Salvador, S.S. Alam, R. Sedlacek, K. Sharma, C. Smith, K. Staes, L. Starrs, F. Sugiyama, S. Takahashi, T. Tanaka, A.W. Trafford, Y. Uno, L. Vanhoutte, F. Vanrockeghem, B.J. Willis, C.S. Wright, Y. Yamauchi, X. Yi, K. Yoshimi, X. Zhang, Y. Zhang, M. Ohtsuka, S. Das, D.J. Garry, T. Hochepied, P. Thomas, J. Parker-Thornburg, A.D. Adamson, A. Yoshiki, J.-F. Schmouth, A. Golovko, W.R. Thompson, K.C.K. Lloyd, J.A. Wood, M. Cowan, T. Mashimo, S. Mizuno, H. Zhu, P. Kasparek, L. Liaw, J.M. Miano, G. Burgio

Research output: Contribution to journalArticlepeer-review


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).

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.

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 languageEnglish
Article number171
JournalGenome biology
Issue number1
Early online date26 Aug 2019
Publication statusPublished - 2019


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

Research Beacons, Institutes and Platforms

  • Global Development Institute
  • National Graphene Institute


Dive into the research topics of 'Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation'. Together they form a unique fingerprint.

Cite this