A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

Abstract

Clustered 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.

Targeted mutagenesis of Δ5 and Δ6 fatty acyl desaturases induce multiplex-mutagenesis and lipogenesis in Atlantic salmon (Salmo salar)

“…Analysis of exonic-part 6 retention in Δ6abc/5 -mutated salmon using RNA-seq data revealed mis-splicing of the Δ6fads2-a transcript resulting in the skipping of exonic part 6 (Figure 2). Exon skipping caused by CRISPR/Cas9-generated mutations was observed previously in both cell lines [31, 32] and genetically modified organisms including zebrafish [33] and salmon [17]. CRISPR induced mis-splicing is mostly caused by one of two mechanisms: i) indels generated by CRISPR-mutation affects the exon-intron boundaries or ii) indels promote exon skipping by disrupting an exon splicing enhancer or introducing an exon splicing silencer within the targeted exon [34].…”

CRISPR/Cas9-mediated editing of GABRR2 gene in RGC-5 cells induces random exon deletion, exon splicing and new exon recruitment

“…CRISPR/Cas9 and its variations provide an efficient and convenient tool for targeted genome editing in mammalian cells, rodent embryo, zebrafish, drosophila, plants and bacteria [1,4,7,8,13,14]. These DNA editing strategies have been widely used for gene knock-out and knock-in, mutation, targeted correction and even chromosome elimination and inversion [15][16][17][18][19], highlighting promising therapeutic applications.…”

“…Repair of the DNA damage often introduces small deletions or insertions that disrupt the target gene and thereby knock out its function. CRISPR/Cas9 has been widely used to generate cell and animal lines with specific gene function being disrupted or knockout [4][5][6][7][8]. Applying the genome surgery to study gene function and to correct disease-associated genetic mutations highlights therapeutic applications in curing genetic disorders.…”

“…CRISPR/Cas9-based genome editing can induce mutations in genome that has high probability to cause exon truncation, deletion or splicing. The accidents that mutations induced exon skipping were usually in the cases of the insertion of a large gene cassette or several nucleotides within exon [7,[9][10][11]. Exon skipping caused by deletion mutations was seldom reported [12].…”

hace1 Influences zebrafish cardiac development via ROS‐dependent mechanisms

“…While effective and efficient, morpholinos can be prone to off-target effects worthy of validation in permanent loss-of-function mutants (Bedell et al, 2011). CRISPR/Cas9 genome editing was employed to target hace1, but the first attempt resulted in no phenotype due to an alternative translation start site (Prykhozhij et al, 2017). The second attempt resulted in identification of an allele with the nonsense-mediated decay activity of 95 %, but the breeding of the F1 mutation carriers often led to large numbers of embryos with abnormalities unlinked to the hace1 mutation, possibly due to off-target mutations or inbreeding (data not shown).…”