Ingrid Hein

October 25, 2016

FLORENCE, Italy — As new technologies are added to the CRISPR–Cas9 gene-editing toolbox, possibilities for more efficient and precise manipulation of DNA open up. However, some scientists worry about unintended and undetected "off-target" DNA alterations.

The inner workings of CRISPR 2.0, including the new Cpf1 "scissors" that can cut DNA in a variety of patterns, were described here at the European Society of Gene and Cell Therapy 2016 Annual Congress by Feng Zhang, PhD, from the Broad Institute and the McGovern Institute for Brain Research at MIT in Cambridge, Massachusetts.

Unlike the previous CRISPR scissors, which cut DNA in a straight line, the new ones can cut in a staggered pattern, increasing precision, which could have a huge effect on the development of treatments for diseases, said Dr Zhang, who is a pioneering designer of CRISPR genome-editing methods.

CRISPR–Cas9 has revolutionized the science of genome editing. It is now possible for any scientist to efficiently and inexpensively conduct gene-editing research.

Previously available approaches to genome modification — such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) — allowed researchers to introduce double-stranded breaks to activate repair pathways, but they were costly and time-consuming.

CRISPR 2.0, which was rolled out over the past year, provides researchers more ways to make genomic edits.

"The way genome editing works is that you cut DNA, then the cell recognizes that the DNA has been cut, and that recognition activates the cell to edit or to repair the genome," Dr Zhang told Medscape Medical News.

The original Cas9 system creates "what we call a blunt cut," he explained. "DNA is double-stranded, so a blunt cut means you're cutting in exactly the same position in both strands. You end up with two blunt ends."

The CRISPR Cpf1 scissors are a "sticky-end cutter," he said. Because they can cut in a staggered pattern, the cut on the top strand of DNA can be in a different place than the cut in the bottom strand.

Different kinds of breaks can be recognized by different DNA repair systems. "Being able to generate a different type of cut gives us the potential to engage other processes. This can make gene-editing more efficient," he pointed out.

Because multiple cuts to genes can be made simultaneously with CRISPR Cpf1, unintentional, or off-target, cuts can result.

Off-Target Cuts Could Cause Unintended Mutations

"Viral-based vectors are still safer than what we can do with CRISPR," said Rik Gijsbers, PhD, from the Laboratory for Vectorology and Gene Therapy at KU Leuven University in Belgium.

For his current research on HIV, CRISPR is not safe enough. "I'm skeptical in the sense that there are still off-targets," he told Medscape Medical News. However, he acknowledged, no tool is 100% safe.

Dr Zhang described a program designed to correct the phrase "twinkle, twinkle big star" to read "twinkle, twinkle little star." Gene-editing tools can simply cut the word "big" out and insert the word "little."

But what if another phrase resides on the same page, such as "Who's afraid of the big bad wolf?" When making the first correction, the editor could unintentionally make a correction to the second phrase, which would end up — incorrectly — as "Who's afraid of the little bad wolf?"

That kind of mistake could create mutations that, for example, could cause cancer, Dr Gijsbers explained. Although the current iteration of CRISPR is good for preliminary animal-model research, "personally, I wouldn't use it for gene therapeutic applications," he said.

Tools must be chosen on the basis of the disease you want to cure or the animal model you want to set up, he pointed out.

"If you don't need to consider off-target effects and you design your research to avoid off-target effects, the system is nice," said Yan Zhou, a PhD candidate at Aarhus University in Denmark.

In his biomedicine lab, CRISPR is being used to research medium-chain acyl-CoA dehydrogenase deficiency (MCADD), a rare genetic disease, Zhou told Medscape Medical News.

"We are using CRIPR on sgRNA to make a break on a gene that has to metabolize fatty acid; if there is a mutation in this gene, the cells cannot metabolize, and the results are mitochondria deficiency," he explained.

CRISPR is very efficient for designing sgRNA. "My lab used TALEN before, but since CRISPR came out, we always use CRISPR; it's much more efficient," Zhou reported. Of course, "you have to be careful."

In a recent study, Dr Zhang and his colleagues identified a new enzyme in the bacterium Leptotrichia shahii that can chop single-stranded RNA (Science. 2016;353:aaf5573). The team refers to the new enzyme — which opens doors for new RNA-targeting tools — as a "class 2 type VI CRISPR–Cas effector C2c2."

Gene Editing Still in its Infancy

The CRISPR system will continue to be developed and improved. "Gene editing is still in its infancy," Dr Zhang explained, pointing out that there is a long way to go in the development of tools for this field.

"We are still trying to develop tools that can overcome some of the fundamental challenges, like how to edit postmitotic cells," he said. And delivery — actually getting modified genes back into humans — is still, for the most part, an unsolved piece of the gene-modification puzzle. But "a lot of people are working on it: drug-delivery people, viral-vector people, nano engineering people," he explained.

As clinical trials that involve humans are approved, continual checks and balances will become important. "We need to double-check ourselves at each juncture to make sure that our decisions are based on high-quality data," Dr Zhang said.

The US Food and Drug Administration invited him to give a lecture at their offices about a month ago. "They know it's an important and complicated technology," he said, and they do not want to make decisions "in a bureau with closed doors."

As they move forward, Dr Zhang and his team are looking more closely at how nature itself solves some of these problems. They are asking whether "we can use some of the things that nature has already developed as inspiration for engineering a new set of tools," he reported.

Dr Zhang said he hopes subsequent developments will be "bio-inspired."

Dr Zhang, Dr Gijsbers, and Mr Zhou have disclosed no relevant financial relationships.

European Society of Gene and Cell Therapy (ESGCT) 2016 Annual Congress. Presented October 20, 2016.

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