Application of CRISPR/Cas9 Technology in Optimizing the Knock-in of Zebrafish Point Mutations

One of the greatest advantages of strategies employing CRISPR/Cas9 is now used in a variety of species is the ability to introduce specific genome modifications. However, introducing defined point mutations has been a significant challenge in some species because it requires some form of homology-directed repair (HDR) or recombination. The zebrafish is one species that has recognized these challenges, despite the broad adoption of CRISPR/Cas9 technology in general and significant advances in many areas.

Zebrafish are frequently used for basic developmental biology research and human disease modeling because of their transparency, fecundity, and availability of well-developed genetic and cell biology tools. For disease modeling, the application of CRISPR/Cas9 to generate missense point mutants of residues conserved between humans and zebrafish may be of particular value, as these types of zebrafish studies are more cost-effective and scalable than other vertebrate model animals, like a mouse. However, this potential will only be realized if the efficiency of current point mutation knock-in strategies is significantly improved.

The first demonstration of small mutational knock-in in zebrafish was a proof-of-concept that such modifications were possible using single-stranded oligodeoxynucleotides (ssODNs), but did not show germline transmission of the introduced mutations. Importantly, the feasibility of using ssODNs to introduce small modifications has actually been demonstrated long before by pioneering work using transcription activator-like effector nucleases (TALENs). However, TALEN-based approaches have not been widely adopted by the field Adoption was used for knock-in generation, possibly because using TALENs was more difficult than producing single guide RNAs (sgRNAs) once CRISPR/Cas9 became available.

Over the past few years, ssODN-based knock-in has advanced in several ways. Although the proportion of correctly modified alleles is low, inserts encoding protein epitope tags such as HA have been successfully introduced. This low-fidelity insertion problem arose in all earlier studies on ssODN-based knock-in, and no systematic attempt to address it has been published. The first report of point mutation insertion and germline transmission in zebrafish is published describing the involvement of modification of the TARDP and FUS genes in amyotrophic lateral sclerosis. The researchers were able to introduce a mutation into the fus gene in 1 of 47 founders using a 33 nucleotide (nt) oligonucleotide and another mutation into 77 using a 100 nt oligonucleotide containing a mutation at the sgRNA site. In the tardp genes of 3 of the founders. None of the successful knock-ins contained indels, but it is unclear whether this is a representative sample from the actual knock-in allelic distribution.

Genetic diseases in humans are often caused by point mutations, but until recently these were modeled in laboratory animals using null mutants of the affected genes, which can lead to phenotypes that are too extreme and may not generalize phenotypes of human patients. Given the tremendous advances in genome editing, the zebrafish model is poised to develop efficient methods for precise point mutation generation.

Generating point mutants using single-stranded DNA oligonucleotides and CRISPR/Cas9 genome editing reagents is an emerging technique in zebrafish and other animal model systems. Single-nucleotide precision for genome editing experiments can be achieved using this technique and will allow generation of specific disease models and precise mutational analysis of biological processes. Despite their apparent promise, point mutation knock-ins remain inefficient in zebrafish, and methods to test their efficiency remain laborious or not readily available to many laboratories, such as individual plasmids by Sanger sequencing and NGS of PCR amplicons clones for sequencing. In an early TALEN-based knock-in study in zebrafish, restriction sites were introduced at specific genomic loci and shown to be digested by the corresponding enzymes, but this approach has not yet been shown to be The CRISPR Approach Efficient Knock-in in Zebrafish.

The proximity of knock-in mutations to the Cas9 cleavage site and antisense asymmetric oligonucleotides were identified as the most effective optimizations. PS modifications also improved knock-in efficiency and improved consistency between different embryos. These optimizations were achieved by AS-PCR analysis and NGS. Restriction sites introduced by silent mutations as part of the knock-in process are also very useful for point mutation genotyping, but only when they are present at a high frequency (e.g. 50%).