Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

Abstract

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.

Fig 1. HDR knockin of point mutations in zebrafish using the CRISPR/Cas9 system with ssODN templates.

Schematic representation of zebrafish tdp-43 (A) and fus (B) and locations of point mutations encoding missense mutations generated by HDR (top). L, nuclear localization sequences; E, nuclear export sequences; RRM, RNA recognition motifs; ZnF, zinc finger motif. Exon coding sequences (middle). Comparisons of zebrafish gRNA target sites with human coding sequences (bottom). Note the high amino acid sequence homologies between human and zebrafish proteins. ALS-causing point mutations (red) encoding missense mutations TARDBP^A382T and FUS^R521H are indicated in the human sequences and analogous point mutations are noted in the zebrafish genes (tardbp^A379T and fus^R536H).

Fig 2. NHEJ reliably generated indels at target site but failed to generate point mutations.

Design of gRNAs targeting our regions of interest in the zebrafish tardbp (A) and fus (B) were generated and co-injected with Cas9 into the one cell stage fertilized egg. gRNA target sites are underlined and PAM sequences are denoted in blue. Germline transmission of mutations was assessed by RFLP analysis. PCR amplicons for tardbp and fus were designed to contain unique restriction enzyme sites (PvuII and MwoI for tardbp and fus respectively) that would fail to cut tardbp^A379T and fus^R536H mutant amplicons. C, Example gel electrophoresis of PCR amplicons of wild type and an example heterozygotes mutant tardbp line. Note the partial cut in our mutant line digested with PvuII D, Sequencing of homozygous F2 larval amplicons for tardbp. E, Example gel electrophoresis of PCR amplicons of wild type and an example heterozygotes mutant fus line. Note the partial cut in our mutant line digested with MwoI F, Sequence results of homozygous F2 larval amplicons for our fus mutant lines. None of our generated lines were single point mutations suggesting that CRISPR/Cas9-medated NHEJ was not a desirable method for generating specific point mutations.

Fig 3. Generation of point mutations encoding tardbp^A379T and fus^R536H was achieved by HDR.

Co-injection of our gRNA, Cas9 and an ssODN template encoding our desired point mutations encoding tardbp^A379T (A) and fus^R536H (E) was made into one cell stage embryos. gRNA target sites are underlined and PAM sequences are denoted in blue. We identified a line in each batch of raised tardbp and fus F0 fish that transmitted an indel, identified by RFLP (B and D) that integrated and transmitted to F1 progeny the tardbp^A379T (C) and fus^R536H (F) missense point mutations identified following sequencing. Corresponding electropherograms of heterozygous F1 progeny indicating our desired point mutations (arrowheads).

Fig 4. HDR ssODN template integration was confirmed by the inclusion of multiple silent around the tardbp^A379T point mutation.

Confirmation of ssODN integration was achieved by co-injection of an ssODN template containing our point mutation of interest and 4 silent point mutations (red nucleotides; A shown for the region flanking the mutation) and was successfully transmitted to F1 progeny; note the double peaks in the electropherogram of heterozygous F1 progeny (top electropherograms; B). We also sequenced the undigested PvuII band and confirmed the integrated template (bottom electropherogram).