What's next for CRISPR? Cas9, Cas3 and beyond

International researchers have collaborated on a new CRISPR-based tool, CRISPR-Cas3 which has been likened to a “DNA shredder with a motor”. This is distinct from CRISPR-Cas9 which has been described as single-sequence targeting ‘scissors’[1].

Read our complete guide to CRISPR Cas9

CRISPR-Cas3 has the ability to erase long stretches of DNA from a targeted site within the genome.  Using this tool, researchers can scan the genome and detect non-coding genetic elements. These elements then act as regulators, controlling the expression of proteins in coding genes, a significant aspect of cell differentiation and sex determination.

CRISPR Cas3 has been likened to a "DNA shredder with a motor"

As Yan Zhang, study leader at the University of Michigan, put it “Cas9 is a molecular scissor that goes where you want it and snips once. Cas3 goes where you want it, travels along the chromosome, and makes a spectrum of deletions tens of kilobases long”[2].  The scale of this tool could give it powerful screening abilities particularly for determining which large areas of DNA are relevant for certain diseases.

Cas3 may also be a significant development for the treatment of viruses, with suggestions that it could find and erase ectopic viruses, such as herpes simplex and hepatitis B. Ailong Ke, professor of molecular biology and genetics said that this tool “can be made to target these viruses very specifically and then erase them very efficiently. In theory it could provide a cure for these viral diseases” [3].

In the latest tests, researchers used Cas3 in HAP1 cells and human embryonic cells, deleting DNA sequences up to 100,000 base pairs.

Read more: How CRISPR immunity and mathematical cryptanalaysis may change the future of personalized medicine

In a recent podcast on humanOS[4] Paul Garofolo, CEO of Locus Biosciences, compared the Cas3 tool to PAC-MAN. As he put it, Cas3 can be used to “to go in [to the cell] and…identify a base pair location. Instead of making a double strand cut [similar to Cas9], it recruits what behaves like a PAC-MAN. It makes a small nick on one strand of the DNA, recruits that PAC-MAN, and it unzips, unwinds and permanently destroys one strand of DNA around the entire genome of a bacteria cell”[5].

"[Cas3] unzips, unwinds and permanently destroys one strand of DNA around the entire genome of a bacteria cell"

Ailong Ke did note that this ability to attack a large section can also be a risk. He pointed out that the team “can’t quite define the deletion boundaries precisely and that is a shortcoming when it comes to therapeutics”[6].  The team have made strides though to understand what the Cas3 has shredded, developing custom informatics pipelines to analyse deep sequencing results and building sensitive, dual-reporter cell lines[7]. Although the researchers do believe there is insight to be gained from “shredding” sections of DNA and analysing what changes occur as a result[8].

Thus far, CRISPR-Cas9 has been the standard CRISPR tool, but researchers have experimented with other forms. Cas12a and Cas12b have been said to be more precise variations which are easier to use. While CasX is a smaller variant which enables easier access routes to cells. The newest development of CRISPR-Cas3 is perhaps only signalling that there is far more that can and will be achieved with these tools.

[1] https://www.cell.com/molecular-cell/fulltext/S1097-2765(19)30217-5

[2] https://www.biotechniques.com/crispr/crispr-upgraded-from-scissors-to-shredder/

[3] https://news.cornell.edu/stories/2019/04/crispr-cas3-innovation-holds-promise-disease-cures-advancing-science

[4] https://blog.humanos.me/antibiotics-are-losing-efficacy-crispr-cas-3-pac-man-may-save-us-guest-paul-garofolo-ceo-locus-bio/

[5] https://blog.humanos.me/antibiotics-are-losing-efficacy-crispr-cas-3-pac-man-may-save-us-guest-paul-garofolo-ceo-locus-bio/

[6] https://news.cornell.edu/stories/2019/04/crispr-cas3-innovation-holds-promise-disease-cures-advancing-science

[7] https://www.uofmhealth.org/news/archive/201904/new-dna-%E2%80%9Cshredder%E2%80%9D-technique-goes-beyond-crispr%E2%80%99s-scissors

[8] https://newatlas.com/crispr-cas3-dna-shredder/59214/