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Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles : a multi-center evaluation
- Source :
- GENOME BIOLOGY, Genome Biology, Vol 20, Iss 1, Pp 1-14 (2019), 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, Eudy, J D, Gan, L, Gao, J, Gonzales, A, Guntur, A R, Guo, H, Harms, D W, Harrington, A, Hentges, K E, Humphreys, N, Imai, S, Ishii, H, Iwama, M, Jonasch, E, Karolak, M, Keavney, B, Khin, N-C, Konno, M, Kotani, Y, Kunihiro, Y, Lakshmanan, I, Larochelle, C, Lawrence, C B, Li, L, Lindner, V, Liu, X-D, Lopez-Castejon, G, Loudon, A, Lowe, J, Jerome-Majewska, L A, Matsusaka, T, Miura, H, Miyasaka, Y, Morpurgo, B, Motyl, K, Nabeshima, Y-I, Nakade, K, Nakashiba, T, Nakashima, K, Obata, Y, Ogiwara, S, Ouellet, M, Oxburgh, L, Piltz, S, Pinz, I, Ponnusamy, M P, Ray, D, Redder, R J, Rosen, C J, Ross, N, Ruhe, M T, Ryzhova, L, Salvador, A M, Alam, S S, Sedlacek, R, Sharma, K, Smith, C, Staes, K, Starrs, L, Sugiyama, F, Takahashi, S, Tanaka, T, Trafford, A W, Uno, Y, Vanhoutte, L, Vanrockeghem, F, Willis, B J, Wright, C S, Yamauchi, Y, Yi, X, Yoshimi, K, Zhang, X, Zhang, Y, Ohtsuka, M, Das, S, Garry, D J, Hochepied, T, Thomas, P, Parker-Thornburg, J, Adamson, A D, Yoshiki, A, Schmouth, J-F, Golovko, A, Thompson, W R, Lloyd, K C K, Wood, J A, Cowan, M, Mashimo, T, Mizuno, S, Zhu, H, Kasparek, P, Liaw, L, Miano, J M & 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, https://doi.org/10.1186/s13059-019-1776-2, Genome Biology
- Publication Year :
- 2019
-
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.
- Subjects :
- Male
ResearchInstitutes_Networks_Beacons/global_development_institute
Mouse
Methyl-CpG-Binding Protein 2
EFFICIENT
0302 clinical medicine
CRISPR-Associated Protein 9
Oligonucleotide
Conditional gene knockout
Medicine and Health Sciences
CRISPR
MODIFIED MICE
Guide RNA
lcsh:QH301-705.5
Conditional
Mice, Knockout
0303 health sciences
EMBRYO
Conditional knockout mouse
Reproducibility
ONE-STEP GENERATION
Regression Analysis
Female
ELECTROPORATION
Transgenesis
CRISPR-Cas9
INTEGRATION
Homology-directed repair
Microinjections
lcsh:QH426-470
INSERTION
Mutagenesis (molecular biology technique)
Computational biology
Biology
Homology directed repair
03 medical and health sciences
National Graphene Institute
Machine learning
Animals
Allele
CRISPR/CAS9
Floxing
Alleles
030304 developmental biology
MUTAGENESIS
Research
Floxed allele
Reproducibility of Results
Biology and Life Sciences
DNA
Long single-stranded DNA
Long single-stranded
Human genetics
lcsh:Genetics
Blastocyst
Global Development Institute
KNOCKOUT
lcsh:Biology (General)
ResearchInstitutes_Networks_Beacons/national_graphene_institute
CRISPR-Cas Systems
Factor Analysis, Statistical
knockout mouse
030217 neurology & neurosurgery
Subjects
Details
- Language :
- English
- ISSN :
- 1474760X
- Database :
- OpenAIRE
- Journal :
- GENOME BIOLOGY, Genome Biology, Vol 20, Iss 1, Pp 1-14 (2019), 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, Eudy, J D, Gan, L, Gao, J, Gonzales, A, Guntur, A R, Guo, H, Harms, D W, Harrington, A, Hentges, K E, Humphreys, N, Imai, S, Ishii, H, Iwama, M, Jonasch, E, Karolak, M, Keavney, B, Khin, N-C, Konno, M, Kotani, Y, Kunihiro, Y, Lakshmanan, I, Larochelle, C, Lawrence, C B, Li, L, Lindner, V, Liu, X-D, Lopez-Castejon, G, Loudon, A, Lowe, J, Jerome-Majewska, L A, Matsusaka, T, Miura, H, Miyasaka, Y, Morpurgo, B, Motyl, K, Nabeshima, Y-I, Nakade, K, Nakashiba, T, Nakashima, K, Obata, Y, Ogiwara, S, Ouellet, M, Oxburgh, L, Piltz, S, Pinz, I, Ponnusamy, M P, Ray, D, Redder, R J, Rosen, C J, Ross, N, Ruhe, M T, Ryzhova, L, Salvador, A M, Alam, S S, Sedlacek, R, Sharma, K, Smith, C, Staes, K, Starrs, L, Sugiyama, F, Takahashi, S, Tanaka, T, Trafford, A W, Uno, Y, Vanhoutte, L, Vanrockeghem, F, Willis, B J, Wright, C S, Yamauchi, Y, Yi, X, Yoshimi, K, Zhang, X, Zhang, Y, Ohtsuka, M, Das, S, Garry, D J, Hochepied, T, Thomas, P, Parker-Thornburg, J, Adamson, A D, Yoshiki, A, Schmouth, J-F, Golovko, A, Thompson, W R, Lloyd, K C K, Wood, J A, Cowan, M, Mashimo, T, Mizuno, S, Zhu, H, Kasparek, P, Liaw, L, Miano, J M & 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, https://doi.org/10.1186/s13059-019-1776-2, Genome Biology
- Accession number :
- edsair.doi.dedup.....289a8204ff6c6cb86fbd647d343d1846