Zebrafish and PCR confirm feasibility of high-throughput editing

CRISPR/Cas9 genome editing is widely used as a tool in genetic studies. Researchers looking at zebrafish have combined this technique with the polymerase chain reaction (PCR) to understand more about the functions of genes in humans. The research is published in Genome Research.

CRISPR/Cas9 zebrafish

Source: NHGRI

CRISPR (clustered, regularly interspaced, short palindromic repeat) describes a pattern of DNA sequences that appears frequently in bacterial DNA, and seems to reflect evolutionary responses to past viral attacks. Cas9 is an RNA-guided nuclease that snips a stretch of DNA in two places. The combination of the two allows researchers to target and delete a particular sequence or to insert a new sequence into DNA. The researchers, led by scientists from the National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH) have developed a platform based on this that allows saturation mutagenesis of the genome and large-scale phenotyping efforts. The platform uses cloning-free single-guide RNA (sgRNA) synthesis, and mutants are identified with fluorescent PCR and multiplexed, high-throughput sequencing.

One way to determine the role of a gene is to knock it out (turn it off or delete it) and see what the effect is on the organism. Using this approach, the team focused on 162 loci in 83 zebrafish genes, with around 50 of these being similar to human genes involved in deafness, and had a 99% success rate for generating mutations. Screening with fluorescent PCR and high-throughput DNA sequencing showed an average germline transmission rate of 28%, which was higher for some genes than others.

“It was shown about a year ago that CRISPR can knock out a gene quickly,” says Shawn Burgess of the NHGRI’s Translational and Functional Genomics. “What we have done is to establish an entire pipeline for knocking out many genes and testing their function quickly in a vertebrate model.”

This translates to six-fold increased efficacy compared with other techniques at homing in on target genes and inserting or deleting specific sequences. Because the approach can be multiplexed, this allows scientists to target and mutate a number of genes at the same time and determine their functions.

Zebrafish are important model organisms in genome research, as they breed rapidly, their embryos are fertilized externally and are transparent, and around 70% of their genes have human counterparts.

“The study of zebrafish has already led to advances in our understanding of cancer and other human diseases,” says NHGRI Director 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.”

This high-throughput approach in animal models could add to knowledge of the functions of many human genes, including disease genes, on a larger and more cost-efficient scale than currently possible, and the team’s next step will be to use the method to knock out about 10 percent of the zebrafish’s roughly 25,000 genes.

“We’ve shown that with relatively moderate resources, you can analyze hundreds of genes,” says Burgess. “On the scale of big science, you could target every gene in the genome with what would be a relatively modest scientific investment in the low tens of millions of dollars.”

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