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. 2017 Jul 5:119:31.9.1-31.9.22.
doi: 10.1002/cpmb.42.

CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens

Sunita S Shankaran  1 , Timothy J Dahlem  2 , Brent W Bisgrove  3 , H Joseph Yost  4 , Martin Tristani-Firouzi  1
Affiliations

CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens

Sunita S Shankaran et al. Curr Protoc Mol Biol. .

Abstract

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.

Keywords: CRISPR/Cas9; FO screen; gene editing; loss-of-function; recessive mutant; zebrafish.

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Figures

Figure 1
Figure 1
CRISPR/Cas9-mediated gene disruption - principle and components. CRISPR/Cas9 is a two component system consisting of a single guide RNA (sgRNA) that includes a 20-nucleotide target sequence specific to the DNA target and a 80-nucleotide Cas9 interaction loop that binds to a recombinant form of Cas9 protein with DNA endonuclease activity. The resulting complex induces a target-specific double-stranded break (DSB). In the absence of donor DNA, the DSB is repaired by the non-homologous end joining (NHEJ) DNA repair pathway, which is an error-prone process. This typically results in insertions/deletions (INDELs), which may lead to disruption of target gene function.
Figure 2
Figure 2
DR274 sgRNA digestion and cloning scheme. A. The DR274 sgRNA plasmid has two BsaI restriction enzyme recognition sites that cut outside the binding site to leave 4bp overhangs. Complementary oligos are ordered, annealed, and then ligated into the overhang sites. B. Optimal CRISPR target site (Red) in exon 2 of flh gene showing PAM sequence (blue) and cut site (green). Design of oligos for cloning flh target site into the BsaI digested DR274 backbone.
Figure 3
Figure 3
Example of DraI restriction enzyme digestion of custom sgRNA constructs showing uncut plasmid and DraI digested plasmid with 1862 bp backbone and 285 bp T7-sgRNA fragment.
Figure 4
Figure 4
Example of sgRNA RNA production as compared DraI digested and uncut plasmid customized DR274 plasmid. sgRNA is approximately 103bp but will not run according to size due to its single stranded nature.
Figure 5
Figure 5
Example of zebrafish embryonic phenotype at 48 hours post fertilization. (A) wild type uninfected embryo. (B) Embryo injected with a mix of sgRNA and Cas9 protein and showing phenotypic differences when compared to wt embryo. This embryo has a shorter body axis and lacks a visible notochord (C) Diagram showing precise injection technique for higher mutation rates. The injection solution shown in red is injected at the junction between the dividing first cell and the yolk.
Figure 6
Figure 6
High Resolution Melt Analysis (HRMA) scientific basis and mutation detection. A. Heterozygous locus with a SNP change/mutation will produce two different PCR duplex amplicon populations. When denatured and re-annealed the parental duplexes will be formed, as will heteroduplexes consisting of one strand of “type” (WT) DNA bound to one strand of “Mutant” DNA. Mismatch at the site of the SNP difference will cause a bulge/bubble in the DNA duplex that lowers the Tm. B. Example HRMA data showing the identification of CRISPR/Cas9 induced mutations as samples that melt sooner than wild type counterparts.
Figure 7
Figure 7
The HRMA results and 15% TBE gel used to analyze PCR products. (A) HRMA showing shifted melting curves with colored lines depicting injected embryos and the grey lines depicting the wild type uninjected control embryos. A greater deflection from the wt grey lines indicates higher mutation rates. (B) 15% TBE gel, which is used to analyze the PCR products and for visualizing DNA duplex formation in the CRISPR/Cas9 injected embryos vs wild type controls. The wt control lane shows a single PCR band while the Mutant embryo lanes show Heteroduplex formation induced by CRISPR injections. Heteroduplexes are indicated by the Red bracket. The Black asterisk * indicates the flh PCR product. The injected embryos were divided into 3 groups based on severity of phenotype penetrance. (C)The band intensity of the flh PCR product was analyzed using Image J software and plotted as a graph. The severity of the flh phenotype correlates directly to decreased levels of the flh PCR product.
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