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. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.

MEKK2 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase gene family involved in regulating multiple MAPK signaling pathways. To elucidate the in vivo function of MEKK2, we generated mice carrying a targeted mutation in the Mekk2 locus. Mekk2 ؊/؊ mice are viable and fertile. Major subsets of thymic and spleen T cells in Mekk2-deficient mice were indistinguishable from those in wild-type mice. B-cell development appeared to proceed similarly in the bone marrow of Mekk2-deficient and wild-type mice. However, Mekk2؊/؊ T-cell proliferation was augmented in response to anti-CD3 monoclonal antibody (MAb) stimulation, and these T cells produced more interleukin 2 and gamma interferon than did the wild-type T cells, suggesting that MEKK2 may be involved in controlling the strength of T-cell receptor (TCR) signaling. Consistently, Mekk2؊/؊ thymocytes were more susceptible than wild-type thymocytes to anti-CD3 MAb-induced cell death. Furthermore, TCR-mediated c-Jun N-terminal kinase activation was not blocked but moderately enhanced in Mekk2 ؊/؊ T cells. Neither extracellular signal-regulated kinase nor p38 MAPK activation was affected in Mekk2 ؊/؊ T cells. In conclusion, we found that MEKK2 may be required for controlling the strength of TCR/CD3 signaling.

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Confocal microscope image of a neonatal Atoh1Cre/+; R26R‐lacZ mouse inner ear crista. Hair cells are labeled with anti‐MYO6 (red). Anti‐lacZ ‐green‐ immunolabeling reveals the hair and supporting cells originated from Atoh1‐lineage. For more information, see the article by Yang et al. in this issue. © 2010 Wiley‐Liss, Inc.