Zebrafish Genome Engineering Using the CRISPR-Cas9 System

Abstract

Geneticists have long sought the ability to manipulate vertebrate genomes by directly altering the information encoded in specific genes. The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease has the ability to bind single loci within vertebrate genomes and generate double-strand breaks (DSBs) at those sites. These DSBs induce an endogenous DSB repair response that results in small insertions or deletions at the targeted site. Alternatively, a template can be supplied, in which case homology-directed repair results in the generation of engineered alleles at the break site. These changes alter the function of the targeted gene facilitating the analysis of gene function. This tool has been widely adopted in the zebrafish model; we discuss the development of this system in the zebrafish and how it can be manipulated to facilitate genome engineering.

Keywords: CRISPR–Cas9; genetic screen; genome editing; knock-in; knockout; zebrafish.

Figure 1. Key Figure, Strategy for zebrafish genome engineering with CRISPR-Cas9

Schematic depiction of the steps in a zebrafish CRISPR experiment. The target is selected (A), the sgRNA is synthesized by in vitro RNA synthesis (B), and injected together with Cas9 protein or cas9 mRNA into the 1-cell stage zebrafish (C). Mutagenesis efficiency is evaluated for each sgRNA using various methods including T7 endonuclease, heteroduplex mobility shift (HMA), and sequencing (D). The phenotype of the F0 animals can also be assessed. Successfully mutagenized F0 animals are raised to adulthood and crossed to WT animals; or crossed to other F0 and the phenotype of the offspring assessed (E). F1 animals are genotyped and sequenced (F) and crossed either to siblings or to independently generated alleles in the same gene (G).

Figure 2. Mutagenesis using transgenic expression of the cas9 holoendonuclease

Transgenesis in zebrafish is generally mediated through the use of the Tol2 transposon carried by a plasmid that is injected at the 1 cell stage. The Tol2 inverted repeats (TIR) are recognized by the Tol2 transposase, generally supplied as injected mRNA, and transposition of the engineered Tol2 transposon occurs into the genome. In one approach (left panel) the two components of the holoendonuclease are expressed from different Tol2 elements. One Tol2 element expresses cas9 using a desired promoter that can be tissue specific, ubiquitously expressed, or driven by a small molecule. The other Tol2 element expresses one or more sgRNAs directed against the target gene or genes. Offspring of a cross between lines carrying these two Tol2 elements will express the holoendonuclease and have a mutated target. These offspring can be identified by markers, not shown, on each Tol2 transposon. For example, the sgRNA expressing Tol2 might be marked with GFP expressed specifically in the lens, while the cas9 expressing Tol2 might be marked with mCherry expressed in the heart. The “lens green, heart red” embryos also express the holoendonuclease. In a separate approach (right panel), both components of the holoendonuclease are expressed from a single transgene and all carriers will also be mutagenized at the target gene or genes. These animals may also be marked as in the left panel.

Figure I. Genomic engineering using ZFN, TALEN, and the CRISPR-Cas9 endonucleases

The three different endonucleases can be designed to site-specifically recognize a single-site in a vertebrate genome. Both ZFN and TALEN are constructed as dimers recognizing two sites in a head-to-head orientation (indicated in green). Modular ZF or TALE domains are used to recognize the specific locus while the FokI endonuclease domain cuts the target. The FokI dimerization interface has been engineered to only allow formation of heterodimers. Expression of both proteins is required to make a functional endonuclease. CRISPR-Cas9 is a complex between the Cas9 protein and a small RNA molecule, the sgRNA. Only the 5′ end of the sgRNA need be changed to change the specificity of this endonuclease. All three endonucleases generate double stranded DNA breaks (DSB) which are repaired by the endogenous DNA repair machinery. This can occur without a template by NHEJ mechanisms or can be templated by exogenous DNA for example in HDR or can incorporate exogenously supplied DNA in a homology independent process. The exact mechanisms of repair are currently being investigated.