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.

Mutations in core components of the spliceosome are responsible for a group of syndromes collectively known as spliceosomopathies. Patients exhibit microcephaly, micrognathia, malar hypoplasia, external ear anomalies, eye anomalies, psychomotor delay, intellectual disability, limb, and heart defects. Craniofacial malformations in these patients are predominantly found in neural crest cells‐derived structures of the face and head. Mutations in eight genes SNRPB, RNU4ATAC, SF3B4, PUF60, EFTUD2, TXNL4, EIF4A3, and CWC27 are associated with craniofacial spliceosomopathies. In this review, we provide a brief description of the normal development of the head and the face and an overview of mutations identified in genes associated with craniofacial spliceosomopathies. We also describe a model to explain how and when these mutations are most likely to impact neural crest cells. We speculate that mutations in a subset of core splicing factors lead to disrupted splicing in neural crest cells because these cells have increased sensitivity to inefficient splicing. Hence, disruption in splicing likely activates a cellular stress response that includes increased skipping of regulatory exons in genes such as MDM2 and MDM4, key regulators of P53. This would result in P53‐associated death of neural crest cells and consequently craniofacial malformations associated with spliceosomopathies.

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Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5’ splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations.