Paper Outlines New Thinking and Knowledge of CRISPR and Off-Target Analysis


CRISPR is widely viewed as the newest, greatest revolution in genetics and medicine, although the breakthrough has come with global controversies and numerous lawsuits. This easy and precise method of editing the genome has the potential to treat previously untreatable genetic diseases and be used to develop new, cutting-edge therapies. But much is still to be learned about the technology, including newer and more accurate targeting enzymes as well as the risks of off-target edits.

When Becky Xu Hua Fu met Brett Robb at a CRISPR conference four years ago, they were both deeply involved in CRISPR, not only working toward characterizing programmable CRISPR nucleases, but the potential off-targets for those genetic edits.

The two published a paper in March in Nature Microbiology on the subject and on Fu’s findings on the complex mechanisms of Cas9 and Cpf1 nucleases, a different set of enzymes than the often-used Cas9.

When they first met, Fu was a post-doctoral researcher in the laboratory of Andrew Z. Fire at Stanford University. She had developed what Robb, scientific director for RNA and genome editing at New England BioLabs, called “a really interesting assay that could basically look at every single mismatch in a guide’s target pair and look at what the impact of cutting with Cas9 would be.”

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Fu and Robb took time to speak with BioSpace about the research and paper.

“We really wanted to understand it as much as Cas9, because it is potentially going to be a really useful tool, as useful as Cas9 has been. And we wanted to learn the basic biology of each of these enzymes and their applications for basic science and research,” Fu said.

Cas9 was the first CRISPR enzyme repurposed for genome editing and still the most broadly used, but it cuts DNA to leave blunt-ended DNA , whereas Cpf1 has staggered cuts. This has implications not only in efficiency, but in off-target edits. One of the advantages of Cpf1 over Cas9 is that Cpf1 processes its own guide RNAs and can potentially be multiplexed to perform multiple editing events.

One of the main points stated in the paper related to this was: “Given that either nicking or double-stranded cleavage of DNA is sufficient to induce a variety of repair and replacement mechanisms in vivo, the resulting profiles illuminate a dual capability of CRISPR-Cas nucleases to initiate genetic change through both types of interaction. These observations challenge binary models in which CRISPR nucleases either cleave or fail to cleave individual targets. Instead, our data indicate a scenario where cleaving and nicking targets can and will coexist in a single experimental or natural condition.”

Robb breaks down the implications of the paper into two camps.

“One is a very practical applied thing, using Cpf1 and Cas9 nucleases in vitro in a cell or manipulating an organism’s DNA in genome editing,” he said.

That means that there may be additional “nicking” activities, or activities that cut only one strand of DNA as compared to both strands.

“It needs to be considered when folks are using these nucleases in vitro for manipulating DNA, in cells for genome editing for organisms,” Robb said.

Currently, researchers deal with potential off-targets by predicting them using computer algorithms. But one of the hopes the authors of the study have is that it will inform updates and potentially new off-target predictive algorithms to incorporate this newly found nicking activity.

“A lot of papers weren’t aware of this intrinsic nicking,” Fu said. “If you talk to a lot of people who use Cas9, they consider anything more than two mutations not even to be an off-target possibility, but our work shows that’s not true.”

The second camp is further describing the basic biology of the CRISPR system and how these proteins work together.

“One of the things we found was Cas9, the cutting portion of this whole process, is actually kind of an anchor for all the rest of the proteins,” Fu said. “They are able to come help with the acquisition of the foreign DNA. It has a lot of implications for how this biological process actually works in its native system, in bacteria, and how these proteins work all together to produce a bacterial system.”

This information becomes even more important when you consider last year’s use of CRISPR-Cas9 by He Jiankui, a Chinese researcher, who modified the DNA of embryos for seven couples. A set of twins whose genomes had been edited was born and another couple is pregnant. This was met with widespread condemnation and investigations, mostly because any edits conducted can be inherited by the children’s children, and because the implications of off-target edits are still unknown.

In fact, on June 3, 2019, researchers at University of California – Berkeley analyzed records in the UK Biobank looking at the history of children born naturally with the same edits He Jiankui made. Jiankui's intention, reportedly, was to provide the children with resistance to HIV. However, the researchers, in analyzing more than 400,000 genomes and associated health records, found that individuals with two mutated copies of the CCR5 gene, the one Jiankui modified, had a significantly higher death rate between the ages 41 and 78 than people with one or no copies. In fact, they had a four-fold increased risk of dying from the flu.

“I think one of the major implications that we’d like people to take away is there are more activities to consider,” Robb said. “The flipside is we also don’t want to scare anybody away from using these technologies—they’re amazing, enabling technologies. We just want folks to be aware of these activities so they can be managed, and this awareness can inform experimental design.”

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