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

0522966 - ÚMG 2020 RIV GB eng J - Článek v odborném periodiku
Sedláček, Radislav - Kašpárek, Petr … celkem 113 autorů
Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.
Genome Biology. Roč. 20, č. 1 (2019), č. článku 171. ISSN 1474-760X. E-ISSN 1474-760X
Grant CEP: GA MŠMT(CZ) LM2015040; GA MŠMT(CZ) ED1.1.00/02.0109; GA MŠMT ED2.1.00/19.0395
Institucionální podpora: RVO:68378050
Klíčová slova: CRISPR-Cas9 * Mouse * Transgenesis * Homology-directed repair * Conditional knockout mouse * Floxed allele * Oligonucleotide * Long single-stranded DNA * Machine learning * Reproducibility
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 10.806, rok: 2019
Způsob publikování: Open access
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1776-2

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.
Trvalý link: http://hdl.handle.net/11104/0307380