CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

Abstract

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).

Keywords: CRISPR; Genetics; Genomics; Model Organisms.

Graphical abstract

Figure 1

Snapshot of the UCSC Genome Browser Website Showing How to Get a DNA Sequence of Interest, Using the Example Shown (Nog2E3)

Figure 2

Results List for sgRNA Oligos in CRISPRscan Highlighted in orange is an example of a sgRNA target site (Nog2E3sg1). The upstream “GG” base pairs are required for in vitro transcription. The combination of CRISPRscan and CFD (Cutting Frequency Determination) scores are designed to help select the best candidate sgRNAs, with high affinity for the targeted sequence and low off-targets prediction. The capital letters in the table represent the sgRNAs sequence; the seed region refers to the 12 bp upstream of the PAM sequence and is crucial for genomic binding and target specificity (Cong et al., 2013). For a detailed explanation and bibliography about sgRNA scores, off-targets, and seed definition, we encourage you to visit the CRISPRscan help page (https://www.crisprscan.org/?page=help).

Figure 3

Stepwise sgRNA Design and Cloning into pDR274

Figure 4

Genotyping PCR Design (A) Target region showing candidate sgRNAs and genotyping primers. (B) Representation of an agarose gel resulting from the genotyping PCR. The WT band present in both control and deletion lane serves as internal control for the PCR and represents the WT DNA sequence. The lower size band can be detected when the deletion occurs, resulting in a smaller DNA molecule. Injected animals, that can be mosaics for the deletion (F0), or fish resulting from crossing with WT animals (F1), heterozygous for the deletion, will generate two bands as genotyping PCR product.

Figure 5

Example of a 1-L Breeding Tank Containing a Separating Barrier between Three Female and Two Male Adult Zebrafish

Figure 6

Pipeline from Microinjection to Establishing a Zebrafish Mutant Line

Figure 7

Two Similar Deletions in a cis-Regulatory Element, Downstream of the Zebrafish nog2 Gene, Achieved through This Protocol (A and B) Representation of two generated genomic deletions of the E3 enhancer (del1 and del2), with sizes of 233 and 227 bp, respectively. (C) One heterozygous male and one female were crossed and their progeny was genotyped to select WT (control) and an heteroallelic combination of del1 and 2 (nog2E3del1/del2). Black arrows represent Fw and Rv primers, used in the genotyping PCR. Corresponding amplified bands are shown, in an agarose gel. Adapted from Amorim et al. (2020).

Figure 8

Representation of a PAGE Gel for Testing a Single sgRNA Genomic DNA from a batch of 8 WT embryos will only contain WT alleles, forming homoduplex DNA (lane 2). DNA from 3 batches of 8 injected embryos, a mixture of “indel” mutations and WT alleles will be present, leading to the formation of heteroduplex DNA, indicating that the injected sgRNA is actively targeting the zebrafish genome. If more than one band is seen on the WT lane, this might be due to the presence of single nucleotide polymorphisms (SNPs) in the targeted genomic sequence. You should only extract and sequence heteroduplex bands that differ from those seen in the WT case.