High-fidelity CRISPR-Cas9 nucleases have no detectable off-target mutations

January 6, 2016
DNA
DNA double helix. Credit: public domain

A new engineered version of the gene-editing CRISPR-Cas9 nuclease appears to robustly abolish the unwanted, off-target DNA breaks that are a significant current limitation of the technology, reducing them to undetectable levels. In their report receiving advance online publication in Nature, Massachusetts General Hospital (MGH) researchers describe how altering the Cas9 enzyme to reduce non-specific interactions with the target DNA may greatly expand applications of the gene-editing technology.

"Our creation of a Cas9 variant that brings off-target effects to levels where we can no longer detect them, even with the most sensitive methods, provides a substantial advance for therapeutic applications in which you want to accurately hit your target without causing damage anywhere else in the genome," says J. Keith Joung, MD, PhD, associate chief for Research and the Jim and Ann Orr MGH Research Scholar in the MGH Department of Pathology, senior author of the Nature paper. "But its impact will also be incredibly important for research applications because off-target effects can potentially confound the results of any experiment. As a result, we envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications."

Used to create targeted DNA breaks at which genetic changes can be introduced, CRISPR-Cas9 nucleases combine a bacterial DNA-cutting enzyme called Cas9 with a short guide RNA sequence that can bind to the target DNA sequence. While easier to use than previous gene-editing tools, CRISPR-Cas9 nucleases have a well-characterized and significant limitation. As described in 2013 studies led by Joung and others, CRISPR-Cas9 nucleases can induce off-target DNA breaks at sites that resemble the on-target sequence. Subsequent investigations by Joung's team and others have reduced but never completely and consistently eliminated these off-target effects.

Joung and his colleagues hypothesized that reducing interactions between Cas9 and the target DNA might more completely eliminate off-target effects while still retaining the desired on-target interaction. The MGH team focused on the fact that certain portions of the Cas9 enzyme itself can interact with the backbone of the target DNA molecule. Pursuing an observation originally made by co-lead author Vikram Pattanayak, MD, PhD, of MGH Pathology, the team altered four of these Cas9-mediated contacts by replacing the long amino acid side-chains that bind to the DNA backbone with shorter ones unable to make those connections. "Our previous work suggested that Cas9 might bind to its intended target DNA site with more energy than it needs, enabling unwanted cleavage of imperfectly matched off-target sites," says Pattanayak. "We reasoned that, by making substitutions at these four positions, we could remove some of that energy to eliminate off-target effects while still retaining full on-target activities."

Co-lead author Benjamin Kleinstiver, PhD, of the MGH Molecular Pathology Unit and Michelle Prew, a research technician in Joung's lab, then tested all 15 possible variants in which any combination of one, two, three or four of those amino acid side-chains were altered and found that one three-substitution and one four-substitution variant appeared to show the greatest promise in discriminating against mismatched target sites while retaining full on-target activities in human cells.

The researchers then more fully characterized the four-substitution variant, which they called SpCas9-HF1 (Sp for the Streptococcus pyogenes bacteria, which is the source of this widely used Cas9, and HF for high-fidelity). They found that this variant induced on-target effects comparable to those observed with the original unaltered SpCas9 when used with more than 85 percent of 37 different guide RNAs they tested. Using GUIDE-Seq, a highly sensitive system Joung's lab developed in 2014 to detect off-target CRISPR-Cas9 effects across the genome, the team found that, while nucleases combining unaltered SpCas9 with seven different guide RNAs induced as many as 25 off-target mutations, use of SpCas9-HF1 produced no detectable off-target effects with six of those guide RNAs and only one off-target site with the seventh. These results were further confirmed using targeted deep-sequencing experiments.

Joung's team also found that SpCas9-HF1 could reduce off-target effects when targeting atypical DNA sites characterized by repeat sequences of one or two nucleotides - sites that are typically subject to many off-target mutations. They developed additional derivatives of SpCas9-HF1 - called HF2, HF3 and HF4 - which could eliminate the few residual off-target effects that persisted with the HF1 variant and a small number of guide RNAs. "If SpCas9-HF1 using a certain guide RNA still produces a handful of off-target effects that are particularly difficult to eliminate, it may be possible to engineer new variants that get rid of even those effects," says Joung, who is a professor of Pathology at Harvard Medical School.

The researchers also showed that SpCas9-HF1, like its naturally occurring counterpart, could be combined with other useful alterations that extend its utility. Previous work from the Joung lab published last summer in Nature had shown that introducing a series of amino acid substitutions could expand the targeting range of unaltered SpCas9. In the current study, the authors show that introducing these same alterations into SpCas9-HF1 also extended the targeting range of the high-fidelity variant. "These results show that these variants should be broadly useful to anyone currently using CRISPR-Cas9 technology," says Kleinstiver. "They can easily be used in place of wild-type SpCas9 and provide a highly effective method for reducing off-target mutations to undetectable levels."

Explore further: New genome-editing platform significantly increases accuracy of CRISPR-based systems

More information: Benjamin P. Kleinstiver et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects, Nature (2016). DOI: 10.1038/nature16526

Related Stories

End of CRISPR-CAS9 controversy

February 10, 2015

The IBS research team (Center for Genome Engineering) has successfully confirmed that CRISPR-Cas9 has accurate on-target effects in human cells, through joint research with the Seoul National University College of Medicine ...

Scientists overcome key CRISPR-Cas9 genome editing hurdle

December 1, 2015

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on "off-target" ...

Recommended for you

6 comments

Adjust slider to filter visible comments by rank

Display comments: newest first

pntaylor
1 / 5 (4) Jan 06, 2016
Sorry, couldn't get past the headline, with the "word" nucleases.
I suspect, this uneducated fool meant to say nuclei.
The English language has certainly gone to Hell, in a hand basket.
I am certainly not an English scholar but what I have seen, in the past 30 years, makes, even me, want to barf.
carthur
5 / 5 (8) Jan 06, 2016
Not 'nuclei', the CRISPR is a 'nuclease'. A nuclease is an enzyme capable of cleaving the bonds between the nucleotide subunits of nucleic acids
pntaylor
not rated yet Jan 06, 2016
Well, I do stand corrected and I thank you, for that. The spelling did look a little funny, to me, but I charged on.
However, although they don't belong here, my comments, on the English language, stand.
RealScience
5 / 5 (2) Jan 06, 2016
@pntaylor: You might also want to ease up on the commas when criticizing the cold-blooded murder of the English tongue.

How about:
I suspect that this uneducated fool meant to say nuclei.
The English language has certainly gone to Hell in a hand-basket.
I am certainly not an English scholar, but what I have seen in the past 30 years makes even me want to barf.
...
Well, I do stand corrected, and I thank you for that. The spelling did look a little funny to me, but I charged on.
However, although they don't belong here, my comments on the English language stand.

(But I agree with you basic premise - the quality of writing, and especially of editing, has diminished in recent years.)
Torbjorn_Larsson_OM
5 / 5 (3) Jan 07, 2016
Yay! Good for those with genetic diseases!

Good also for me, I can start dreaming of having my 2nd hearth and eyes in the back. Well, maybe it is too late (and complicated) to make developmental changes... Longevity gene treatment, then.

Oy vey, there will be a looooong ethical debate after this one. Designer kids _and_ designer parents,

[My 2c:

Many genes will not fixate in the population because the treatments are costly. But we risk it because a way to keep costs down is to go for popular changes.

Personal risk is on personal responsibility though. I can sleep well. Hey, what about alleles for better sleep!]
barakn
5 / 5 (3) Jan 07, 2016
Maybe pntaylor talks like William Shatner, in which case the extra commas might be warranted.

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.