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Abstract
ABSTRACTClustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.
Highlights
IntroductionThe original demonstrations that clustered regularly interspaced palindromic repeats (CRISPR) RNA with tracrRNA or engineered single-guide RNA (sgRNA) created from CRISPR RNA and tracrRNA can program Cas nuclease specificity in vitro (Gasiunas et al, 2012; Jinek et al, 2012) and in cells (Cho et al, 2013; Cong et al, 2013; Mali et al, 2013) have opened the floodgates for work on genome editing, one application of which is to generate inactivating mutations in protein-coding genes either by targeting single sgRNA sites to create frameshifts or inducing larger gene deletions with multiple sgRNAs. When introduced into cells, Cas9/ sgRNA complexes induce double-strand breaks at defined sequences, which are typically repaired by the error-prone nonhomologous end-joining or microhomology-mediated end-joining DNA repair pathways resulting in small insertions or deletions (indels) (Rodgers and Mcvey, 2016)
Screening of F1 mutation carriers and identifying their mutations Given zebrafish generation time of about 3 months, it takes 3-4 months to obtain adult founder (F0) animals, some of which carry from one to several mutations distributed in a mosaic fashion, which makes them difficult to use for consistent mutational analysis experiments
heteroduplex mobility assay (HMA) protocols have been presented for genotyping zebrafish injected with zinc-finger nucleases (Chen et al, 2012), transcription activator-like effector nucleases (TALENs) (Ota et al, 2013) and clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 (Ota et al, 2014) reagents, but these papers did not describe their HMA procedures in sufficient detail or focused on mutation detection in very small PCR products
Summary
The original demonstrations that CRISPR RNA with tracrRNA or engineered single-guide RNA (sgRNA) created from CRISPR RNA and tracrRNA can program Cas nuclease specificity in vitro (Gasiunas et al, 2012; Jinek et al, 2012) and in cells (Cho et al, 2013; Cong et al, 2013; Mali et al, 2013) have opened the floodgates for work on genome editing, one application of which is to generate inactivating mutations in protein-coding genes either by targeting single sgRNA sites to create frameshifts or inducing larger gene deletions with multiple sgRNAs. When introduced into cells, Cas9/ sgRNA complexes induce double-strand breaks at defined sequences, which are typically repaired by the error-prone nonhomologous end-joining or microhomology-mediated end-joining DNA repair pathways resulting in small insertions or deletions (indels) (Rodgers and Mcvey, 2016). Recent evidence suggests the presence of upstream open reading frames (uORFs) in approximately half of mammalian mRNAs (Ingolia et al, 2011), which means that translation of main ORFs in these mRNAs requires translation re-initiation. uORFs typically reduce
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