Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease

Abstract

The study of model organisms has revolutionized our understanding of the mechanisms underlying normal development, adult homeostasis, and human disease. Much of what we know about gene function in model organisms (and its application to humans) has come from gene knockouts: the ability to show analogous phenotypes upon gene inactivation in animal models. The zebrafish (Danio rerio) has become a popular model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. The RNA-guided CRISPR/Cas9-mediated targeted mutagenesis approaches have provided powerful tools to manipulate the genome toward developing new disease models and understanding the pathophysiology of human diseases. CRISPR-based approaches are being used for the generation of both knockout and knock-in alleles, and also for applications including transcriptional modulation, epigenome editing, live imaging of the genome, and lineage tracing. Currently, substantial effort is being made to improve the specificity of Cas9, and to expand the target coverage of the Cas9 enzymes. Novel types of naturally occurring CRISPR systems [Cas12a (Cpf1); engineered variants of Cas9, such as xCas9 and SpCas9-NG], are being studied and applied to genome editing. Since the majority of pathogenic mutations are single point mutations, development of base editors to convert C:G to T:A or A:T to G:C has further strengthened the CRISPR toolbox. In this review, we provide an overview of the increasing number of novel CRISPR-based tools and approaches, including lineage tracing and base editing.

Keywords: CRISPR/Cas9; Cas12a (Cpf1); base editors; disease models; lineage tracing; zebrafish.

FIGURE 1

Overview of mutant identification method using fragment analysis approach. (A) Gene specific primers are designed covering the target site (amplicon size ranging 200–300 bp). Gene-specific forward primer contains M13F sequence at the 5′, and reverse primer has PIG-tail sequence at 5′ end. PCR is performed using gene specific primer set, and a third primer with M13F sequence labeled with FAM, resulting amplicons are fluorescently labeled. (B) Fluorescently labeled primers are mixed with size standard (e.g., ROX-400), and run on ABI capillary sequencer, and data is analyzed using gene mapper software. The output will have the size of amplicon, wild type allele will have only one size, while mutant allele will have two different sizes. The indel size can be determined by comparing the size of two alleles (WT vs. mutant).

FIGURE 2

Comparison between Cas9 and Cas12a. (A) Screenshot of UCSC genome browser tracks showing predicted target sequences in tyr gene for CjCas9, and SpCas9. The targets for both Cas9 nucleases are enriched in coding exons, (B) while Cas12 target sequences are enriched mostly in intronic sequences. (C,D) Comparison of Cas12a and Cas9, Cas12 a generates a staggered cut, Cas9 induces a blunt end cut.

FIGURE 3

Single nucleotide substitution using base editors. (A) Cytidine deaminase fused to nickase Cas9 converts cytosine to thymine to guanine to adenine within a targeting window. (B) Adenine base editor converts adenine to inosine that is recognized as guanine during DNA replication or repair thus converting A to G or C to T. (C) In vivo substitution of adenine to guanine using ABE7.10 base editor in zebrafish. sgRNA targeting cx35.4 gene was injected in 1-cell stage zebrafish embryos, DNA from a pool of four injected embryos was sequenced, and 20% of the clones carrying the desired A to G substitution.