A CRISPR approach

NHGRI scientists combine CRISPR/Cas9 and zebrafish for high-throughput, multiplexed gene editing

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BETHESDA, Md.—Researchers at the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health, shared in a recent study that the CRISPR/Cas9 gene-editing technology is six times more effective than other techniques at targeting genes and inserting or deleting specific sequences. The paper, “High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9,” appeared online in June and will be published in the July issue of Genome Research.
CRISPR, or clustered, regularly interspaced, short palindromic repeat, refers to a pattern of DNA sequences that frequently appear in bacterial DNA, sequences that are thought to reflect evolutionary responses to past viral attacks. The Cas9 protein is a nuclease, an enzyme that cuts DNA in two places. This method offers significant specificity and utility in gene editing, allowing scientists to isolate genes to determine their function and the effects of knocking them out.
“It was shown about a year ago that CRISPR can knock out a gene quickly,” said Dr. Shawn Burgess, a senior investigator with NHGRI’s Translational and Functional Genomics Branch and head of the Developmental Genomics Section. “What we have done is to establish an entire pipeline for knocking out many genes and testing their function quickly in a vertebrate model.”
The team used the CRISPR/Cas9 method to target 162 locations in 83 zebrafish genes, resulting in mutations in 82 of the 83 targeted genes. After screening embryos via fluorescent polymerase chain reaction and high-throughput DNA sequencing, the scientists found that mutations were passed on to the next generation in 28 percent of cases.
“Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts,” the authors noted in the abstract of the study, adding that “predicted off-target mutagenesis is of low concern for in-vivo genetic studies.”
In addition, this method has potential as an effective “multiplexed” approach as well, in terms of targeting and mutating multiple genes at the same time. While that might seem like the researchers are making things more difficult for themselves, in trying to track the effects of multiple mutations, Burgess says the opposite is true.
“It seems more complicated, but it actually greatly simplifies our analysis,” he explains. “If you are testing five genes simultaneously, it reduces your animal husbandry (the largest source of labor) five-fold; instead of each fish being genotyped for one gene, they are genotyped for five. Zebrafish typically generate one to 200 embryos in a cross, so there are sufficient numbers of offspring that all genes will segregate independently and can be tested individually. If there are also genetic interactions between mutations, they can also be robustly detected by combinatorial genetics. By reducing the animal husbandry, we can rely more on aspects of the research that can be automated and multiplexed easily. This gives us much greater ability to screen large numbers of mutants for the desired phenotypes.”
Zebrafish have long been an organism of interest for laboratory researchers. The fish are both resilient and extremely prolific, as a female can produce as many as 200 eggs at a time. One of the most appealing aspects about this species is the fact that approximately 70 percent of zebrafish genes appear to have human counterparts.
“The study of zebrafish has already led to advances in our understanding of cancer and other human diseases,” said NHGRI Director Dr. Eric Green. “We anticipate that the techniques developed by NHGRI researchers will accelerate understanding the biological function of specific genes and the role they play in human genetic diseases.”
Burgess says that for him, this research “modernizes vertebrate genetics and fuses it with genomics.”
“Because of big genome projects, we have a well-documented list of genes in the genome. In order to screen through a subset of those genes for desired phenotypes, we no longer have to either rely on random mutagenesis and massive screening efforts, which are difficult in vertebrate models, or small-scale gene targeting approaches, where only a few genes can be tested one by one. It is possible to screen through hundreds or thousands of genes in zebrafish now with a comparatively modest workforce. We hope to knockout and test roughly 2,000 zebrafish genes in the next three to five years,” he says.
Burgess tells DDNews that his lab is currently pursuing a trio of projects. First off, having identified the known and suspected genes that lead to deafness, the team wants to know “how often we can robustly model a human disease using zebrafish.” The second project will focus on identifying the genes that allow zebrafish to regenerate hair cells in the wake of hearing damage to see if similar regeneration can be reactivated in mammals. Burgess says the third project will focus on “targeting the ‘druggable genome’ to functionally annotate what all kinases, [G-protein-coupled receptors] and channels do in a vertebrate genome.”

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