By: David Shifrin, PhD
Science Writer, Filament Life Science Communications

Determining whether a genetic mutation is causative of a specific phenotype or disease is a significant challenge in both basic research and clinical practice. Now, a team of researchers from the National Human Genome Research Institute (part of the NIH) has developed a method to make it a bit easier.

Guarav Varshney, Shawn Burgess and colleagues used the CRISPR/Cas9 gene editing system to create and screen large numbers of mutations in zebrafish. We have talked about CRISPR on this blog before: for a review, see the post CRISPR: treating the genetic underpinnings of disease with a bacterial “immune system”?

Briefly, CRISPR/Cas9 is part of a bacterial adaptive immunity system. Bacteria contain short sequences of DNA (called spacers) that match DNA from invading viruses. These spacer sequences are interspersed between clustered regularly interspaced short palindromic sequences – CRISPRs. When viral DNA is detected, the bacterium transcribes the full sequence and then cuts it to free the spacer RNA (now called crRNAs or, in a lab setting, guide RNAs). These crRNAs then guide proteins to cut viral DNA, rendering it harmless. This process has been adapted in the lab to allow for extremely efficient mutation, repair and editing of genes.

For another look at the system (which includes an extremely basic introduction to genetics), see “Genome Editing with CRISPR-Cas9” on YouTube.

In their Genome Research article called “High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9,” Varshney et al used CRISPR to induce mutations in the zebrafish genome. This is a cleaner process than previous techniques such as chemical mutagenesis (which are somewhat unpredictable and have off-target effects) and zinc finger nucleases (which are somewhat effective but relatively inefficient). The team targeted 83 genes, designing two distinct guide RNA per gene. Having two guide RNAs increased the chance of editing any given gene, since two separate sequences would be targeted.

Remarkably, the researchers found that not only was the efficiency of their CRISPR system quite high in terms of hitting many of the targeted genes, but also the heritability of mutations was quite high. Roughly half of the mutated parent fish showed mutations that were in the germline. Even more importantly, the high efficiency of the CRISPR system meant that the team only needed to screen ~7 fish for every gene, rather than the ~60 required when zinc finger nucleases are used. Thus, using CRISPR to screen mutations in genes that are homologous to potentially relevant human genes requires a fraction of the resources needed for previous technologies.

Another exciting point about CRISPR is that the system works such that more than one gene can be targeted at a time. In zebrafish, this is helpful because of frequent functional redundancy in genes. Knocking out one gene is not always sufficient to induce a phenotype. The authors of this paper showed that, indeed, “multiplex” targeting was effective. Another benefit is that this further reduces the number of animals required, leading to faster validation of human disease genes.

A final improvement offered by CRISPR in this study was the ability to screen for many of the potential phenotypes in offspring from the first generation following mutation. Other methods often require multiple crossbreeding steps, which obviously increases the time to get any worthwhile results.

As validation, the authors tested CRISPR on several genes directly linked to human disease; in this case, deafness. Targeting three of these eight genes (such as those involved in Usher Syndrome) led to hearing-loss phenotypes. While a 100% success rate would be ideal, this still represents a good step forward. Factors aside from the use of CRISPR could also be responsible for the lack of phenotype in the other five mutants.

Interestingly, another paper came out about the same time in Nature, in which the authors describe their engineering of a new form of Cas9 nuclease. This protein is required for the cutting of target DNA. As the authors point out, the natural version has limitations that make it “difficult to target double-stranded breaks with the precision that is necessary for various genome-editing applications.”

To get around this problem, the team led by Benjamin Kleinstiver and J. Keith Joung engineered new versions of Cas9 that carried out more effective and specific targeting in both zebrafish and human cells. Though a long way off, the authors suggest that their findings mean that engineering many such Cas9 variants with increased specificity is possible.

Together, the improvements described in these papers could lead to significant advances on both the analysis and therapeutic sides. One could envision a situation where a new candidate variant is brought to the lab for screening in an animal model, a process accelerated by the use of CRISPR. Once confirmed as disease-causing, the lesion could be targeted for repair by a CRISPR-Cas9 system using the engineered Cas9 nuclease. If the system can indeed make validation and therapy faster, cheaper, and more specific, what’s not to like?

Thu. July 9, 2015