Precision Medicine, CRISPR, and Genome Engineering Moving from Association to Biology and Therapeutics

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5.3 Genome Editing in Zebrafish

For many years, researchers have focused on developing methods to rapidly and

efficiently edit the zebrafish genome. These efforts came to fruition about 10 years

ago with the development of programmable site-specific nucleases; first ZFNs and

then TALENs and most recently with the programmable bacterial endonuclease

Cas9 [ 37 ]. These methods, particularly CRISPR/Cas9, which is the easiest and most

efficient strategy, are transforming biological research enabling new discoveries.

They have also led to the disturbing discovery that morpholinos (the previously

accessible and straightforward gene knockdown tool in zebrafish) caused pheno-

types that were often not recapitulated in genetic knockouts [ 38 ]. This finding may

be partially explained by genetic compensation that can occur in response to genetic

mutations [ 39 ]. Nevertheless, new guidelines in the zebrafish field now recommend

morpholino phenotype corroboration that includes genome editing [ 40 , 41 ].

In zebrafish, CRISPR/Cas9 genome editing has already been used in many dif-

ferent ways; to generate targeted knockouts and knockins, in transgenic strains

expressing Cas9 in specific tissues, and for forward genetic screens to identify new

players in different biological processes. Excellent and comprehensive recent

reviews summarize the research underlying the uses and advances of genome edit-

ing in zebrafish [ 37 , 42 , 43 ]. The most straightforward and successful strategy so far

is the use of CRISPR/Cas9 genome editing to generate gene knockouts. This

approach can be cheaply and efficiently done in all labs competent in basic molecu-

lar biology techniques. As an example, I highlight our approach generating knock-

out models of the MCU.

Making global knockouts in zebrafish using CRISPR/Cas9 is simple and

inexpensive.

Briefly, our strategy is as follows. More experimental details can be found in the

excellent protocols widely used by the zebrafish community [ 44 , 45 ].

1. Multiple databases exist for the selection of the 20nt complementary base pair-

ing sequence that directs Cas9 activity to a specific gene [ 37 ]. This 20nt sequence

is part of the single guide RNA (sgRNA) that also contains a 42nt Cas9-binding

hairpin and a 40nt terminator sequence. We use at least two different programs

to identify target sequences and pick sequences that are predicted in more than

one program to not have off target sites. We also try to choose sequences within

an exon near the 5′ end of the gene.

2. To generate the sgRNA, we subclone our target sequence into the pT7-gRNA

from the Chen and Wente lab [ 46 ]. This plasmid is designed for the rapid and

efficient cloning of target sequence into the sgRNA backbone. Similarly we have

synthesized Cas9 mRNA using the version developed by this same group that

has nuclear localization sequences at both the amino and carboxyl termini and

has further been optimized for expression in zebrafish [ 46 ].

3. After in vitro transcription of both the Cas9 mRNA and the sgRNA and subse-

quent purification, we inject these into fertilized zebrafish eggs at the 1–2 cell

stage. We do several experiments in which we first optimize the concentration of

sgRNA to minimize lethality, but maximize mutagenesis.

S.E. Brockerhoff