Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

Abstract

Background Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies—definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. Results We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes. Conclusions Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice.

Strategies to Target ADAM17 in Disease: From Its Discovery to the iRhom Revolution

“…In addition, iRhom1 and 2 double knockout mice die in utero at E9.5, rather than perinatally, as it is shown by Li et al This suggests that the function of iRhom1 and 2 is not fully redundant, and that they must have additional functions to guiding ADAM17 maturation. The discrepancy between these two mouse models may arise from the strategy used to ablate iRhom1 and 2, as has been reported by Hosur et al [67]. This work demonstrated that the complete deletion of iRhom1 coding sequence leads to the severe phenotype reported by Christova et al In contrast, the strategy used by Li et al of deleting the sequence spanning from exon 4 to 11, where the canonical starting codon resides, leads to the expression of a truncated form of iRhom1 that originates from an alternative starting codon.…”

Highly efficient manipulation of nervous system gene expression with NEPTUNE

“…In mice, genetic background can alter phenotype penetrance, but even in a defined genetic background penetrance can be variable ( Dickinson et al, 2016 ). Additionally, genetic “knockouts” generated by using different strategies (deletion of start exons, all exons, or CRISPR/Cas9 deletion of a few exons) can yield different phenotypes ( Hosur et al, 2020 ). CRISPR/Cas9-mediated gene editing can lead to exon skipping and hypomorphism, neomorphism, or gain of function of the targeted gene, leading to unexpected phenotypes that do not reflect loss of function ( Anderson et al, 2017 ; Chen et al, 2018 ; Uddin et al, 2015 ).…”

Electroporation-Mediated Genome Editing of Livestock Zygotes

“…Gene knockouts using the NHEJ pathway have been the most successful type of embryo-mediated genome edit, to date, and there are several experiments documenting very high rates of bi-allelic mutation using electroporation. Although it should be noted that gene compensation through exon skipping has been observed to reinitiate transcription and translation, which can result in partial gain-of-function alleles rather than the predicted nonsense or missense alleles (Lalonde et al, 2017;Smits et al, 2019;Hosur et al, 2020) When the editing reagents are working well and producing 100% bi-allelic knockouts, transferring edited embryos carries little downside. However, if rates decrease below this, the probability of transferring mosaic, hemizygous, or wild type animals increases.…”