Sharpening CRISPR-Cas’s Aim

Shortening guide RNAs markedly improves gene-editing tool

Image: NIH's National Human Genome Research Institute.

A simple adjustment to a powerful gene-editing tool improves its precision, Harvard Medical School researchers at Massachusetts General Hospital report.

In a paper published in Nature Biotechnology, the scientists have shown how adjusting the length of guide RNAs in synthetic enzymes called CRISPR-Cas RNA-guided nucleases can substantially reduce off-target DNA mutations, a limitation the team revealed just last year.

“Simply by shortening the length of the guide RNA targeting region, we saw reductions in the frequencies of unwanted mutations at all of the previously known off-target sites we examined,” said J. Keith Joung, HMS associate professor of pathology at Mass General and senior author of the paper. “Some sites showed decreases in mutation frequency of 5,000-fold or more, compared with full-length guide RNAs. Importantly, these truncated guide RNAs—which we call tru-gRNAs—are just as efficient as full-length gRNAs at reaching their intended target DNA segments.”

Last year two groups reported their success in using a tool borrowed from a bacterial immune system called Cas, short for CRISPR-associated systems, which in turn stands for Clustered Regularly Interspaced Short Palindromic Repeats. In bacteria the Cas9 enzyme system uses short stretches of RNA to target and then cut invading viral DNA. Scientists have customized this system to work in human cells, creating an RNA-guided editing tool that allows them to integrate DNA changes into the genomes of living cells.

Later last year Joung’s team found that in human cells, CRISPR-Cas RNA-guided nucleases could also cause mutations in DNA sequences with differences of up to five nucleotides from the target, which could seriously limit the proteins’ clinical usefulness. The team followed up with a hypothesis that could seem counterintuitive: Shortening the gRNA segment might reduce off-target mutations.

“Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity,” Joung explained. “That led us to wonder whether removing these nucleotides could make the system more sensitive to mismatches in the remaining sequence.”

The CRISPR-Cas RNA-guided nucleases most widely used by researchers include a 20-nucleotide targeting region within the gRNA. To test their theory, the team constructed RNA-guided nucleases with progressively shorter gRNAs. They found that while gRNAs with targeting segments of 17 or 18 nucleotides were at least as efficient as full-length gRNAs in reaching their targets, those with 15- or 16-nucleotide targeting segments had reduced or no targeting activity. Subsequent experiments found that 17-nucleotide truncated RNA-guided nucleases efficiently induced the desired mutations in human cells, with greatly reduced or undetectable off-target effects, even at sites with only one or two mismatches.

“While we don’t fully understand the mechanism by which tru-gRNAs reduce off-target effects, our hypothesis is that the original system might have more energy than it needs, enabling it to cleave even imperfectly matched sites,” Joung said. “By shortening the gRNA, we may reduce the energy to a level just sufficient for on-target activity, making the nuclease less able to cleave off-target sites. But more work is needed to define exactly why tru-gRNAs have reduced off-target effects.”

Joung’s team has incorporated this capability for finding tru-gRNA target sites into ZiFiT Targeter, a freely available software package designed to identify potential target sites for several DNA-editing technologies. 

The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM088040 and P50 HG005550, and the Jim and Ann Orr MGH Research Scholar Award.

Adapted from a Mass General news release.