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Fig. 5 | Genome Biology

Fig. 5

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

Fig. 5

An “incomplete protein-coding CDS” approach to identifying on-target unintended transcription and translation reinitiation. a Bioinformatics analyses of mouse (left) and human (right) protein-coding genes with incomplete 5′ CDSs and incomplete 3′ CDSs reveals that ~ 50% of genes are potentially capable of generating shorter transcripts and truncated proteins. The physiological significance of these incomplete CDSs is unclear. However, it is yet to be determined whether gene-targeting strategies must be designed to ensure the deletion of these incomplete, yet existing coding sequences that are not degraded by NMD. Notably, a 3′ incomplete CDS is a protein-coding transcript which is missing the stop codon due to incomplete evidence and a 5′ incomplete CDS is a protein-coding transcript which is missing the start codon due to incomplete evidence. b The various transcripts of the mouse Rhbdf1 gene listed in Ensembl. In this scenario, the definitive-null design, rather than either the targeted KO-first or CRISPR/Cas9-mediated deletion of exon(s) containing the start codon, would prevent expression of a short 5′ incomplete Rhbdf1-206 transcript. c CRISPR/Cas9-mediated frameshift mutation failed to induce complete deficiency of BAZ2A in HAP1 cells; however, based on the presence of 5′ and 3′ incomplete CDSs (arrows), BAZ2A is likely to generate truncated proteins upon targeted KO-first- or CRISPR/Cas9-induced on-target mutagenesis. d Bioinformatics analyses of 3674 conditional-ready mouse strains identified that nearly 55% of conditional-ready genes generate incomplete 5′ and 3′ CDSs. e Schematic of Bbs5 KO-first allele in which the “lacZ reporter gene and neomycin resistance gene” flanking FRT recognition sites is placed downstream of the third exon of the gene, and exons 4 and 5 are floxed by loxP sites. Following Cre-mediated recombination, despite missing exons 4 and 5, Bbs5 5′ incomplete CDS 203 may generate an N-terminally truncated protein. f Bioinformatics analyses of 410 lethal IMPC mouse strains identified that 166 essential genes that generate incomplete 5′ and 3′ CDSs. g Schematic of Anapc4 KO-first allele in which the “lacZ reporter gene and neomycin resistance gene” flanking the FRT recognition sites is placed downstream of the second exon of the gene, and exon 3 is floxed by loxP sites. Although Cre recombination deletes exon 3, translation reinitiation from incomplete CDSs 209 and 211 may rescue the lethal phenotype of Anapc4 homozygous-null mice. h Analyses of 198 subviable IMPC mouse strains identified 81 genes generating incomplete 5′ and 3′ CDSs. i The targeted KO-first strategy in Dnmt3a results in conflicting phenotypes. Whereas the tm1a KO-first allele generates homozygous-viable mice, the tm1b allele generates homozygous subviable mice (top). Multiple protein-coding transcripts of Dnmt3a are shown. The Dnmt3a 207 protein-coding transcript has incomplete 5′ and 3′ CDSs (arrow) (bottom)

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