The genome editing system CRISPR has become a hugely important tool in medical research, and could ultimately have a significant impact in fields such as agriculture, bioenergy, and food security.
The targeting system can travel to different points on the genome, guided by a short sequence of RNA, where a DNA-cutting enzyme known as Cas9 then makes the desired edits.
However, despite the gene-editing tool’s considerable success, CRISPR-Cas9 remains limited in the number of locations it can visit on the genome.
That is because CRISPR needs a specific sequence flanking the target location on the genome, known as a protospacer adjacent motif, or PAM, to allow it to recognize the site.
For example, the most widely used Cas9 enzyme, Streptococcus pyogenes Cas9 (SpCas9), requires two G nucleotides as its PAM sequence, significantly restricting the number of locations it can target, to around 9.9 percent of sites on the genome.
As yet, there are only a handful of CRISPR enzymes with minimal PAM requirements, meaning they are able to target a wider range of locations.
Now researchers at the MIT Media Lab and the Center for Bits and Atoms, led by Joseph Jacobson, a professor of media arts and sciences and head of the Molecular Machines research group, have discovered a Cas9 enzyme that can target almost half of the locations on the genome, significantly widening its potential use. They report their findings in Science Advances Oct. 24.
“CRISPR is like a very accurate and efficient postal system, that can reach anywhere you want to go very precisely, but only if the ZIP code ends in a zero,” Jacobson says. “So it is very accurate and specific, but it limits you greatly in the number of locations you can go to.”
To develop a more general CRISPR system, the researchers implemented computational algorithms to conduct a bioinformatics search of bacterial sequences, to determine if there were any similar enzymes with less restrictive PAM requirements.
To carry out the search, the researchers developed a data analysis software tool, which they called SPAMALOT (Search for PAMs by Alignment of Targets).
This revealed a number of interesting possible enzymes, but no clear winner. So the team then built synthetic versions of the CRISPRs in the laboratory, to evaluate their performance.
They found that the most successful enzyme, a Cas9 from Streptococcus canis (ScCas9), was strikingly similar to the Cas9 enzyme already widely used, according to co-lead author Pranam Chatterjee, a graduate student in the Media Lab, who carried out the research alongside fellow graduate student Noah Jakimo.
“The enzyme looks almost identical to the one that was originally discovered … but it is able to target DNA sequences that the commonly used enzyme cannot,” Chatterjee says.
Rather than two G nucleotides as its PAM sequence, the new enzyme needs just one G, opening up far more locations on the genome.
This should allow CRISPR to target many disease-specific mutations that have previously been out of reach of the system.
For example, a typical gene is around 1,000 bases in length, giving researchers a number of different locations to target if their aim is to simply knock out the entire gene, Jacobson says.
However, many diseases, such as sickle cell anemia, are caused by the mutation of a single base, making them much more difficult to target.
“Base editing is not just a matter of hitting that gene anywhere over the 1,000 bases and knocking it out; it is a matter of going in and correcting, in a very precise way, that one base that you want to change,” Jacobson says.
“You need to be able to go to that very exact location, put your piece of CRISPR machinery right next to it, and then with a base editor — another protein that’s attached to the CRISPR — go in and repair or change the base,” he says.
The new CRISPR tool could be particularly helpful in such applications.
“We are excited to get ScCas9 into the hands of the genome editing community and receive their feedback for future development,” Chatterjee says.
The researchers very elegantly took advantage of the natural evolution of Cas9 sequences in Streptococcus bacteria in order to identify the new Cas9 protein, which will be a powerful tool for genome editing, says Jean-Paul Concordet, a CRISPR specialist at the National Museum of Natural History in France, who was not involved in the research.
“The amino acid sequence of ScCas9 is very closely related to that of SpCas9, so the anticipation is that it will also prove very easy to produce in recombinant form and can directly benefit from all the developments made for SpCas9,” Concordet says.
“In addition, ScCas9 works with the same guide RNAs as SpCas9, so it will be possible to use synthetic guide RNAs that are readily available from different companies,” he says.
The researchers are now hoping to use their technique to find other enzymes that could expand the targeting range of the CRISPR system even further, without reducing its accuracy, according to Jacobson.
“We feel confident of being able to go after every address on the genome,” he says.
The Latest on: CRISPR
via Google News
The Latest on: CRISPR
- CRISPR Stocks: The Big Analyst Call Destroying CRSP, NTLA, EDIT Stocks Todayon April 16, 2021 at 12:51 pm
A dramatically bearish analyst take has CRISPR stock Editas (NASDAQ:EDIT) down 15% in trading today, but it’s not ...
- CRISPR base editor treats premature-aging syndromeon April 16, 2021 at 7:10 am
CRISPR-mediated adenine base editor (ABE) to repair mutations of the Hutchinson–Gilford progeria syndrome (HGPS or progeria), attenuate symptoms, and extend lifespan of mice (Fig. 1 ), 1 representing ...
- CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cellson April 16, 2021 at 2:27 am
Incidence trends and survival prediction of hepatoblastoma in children: a population-based study. Cancer Commun (Lond). 2019;39:62. Zhang X, Song M, Kundu JK, Lee MH, Liu ZZ. PIM kinase as an ...
- New CRISPR Technology Offers Unrivaled Control of Epigenetic Inheritanceon April 15, 2021 at 5:45 pm
Scientists have figured out how to modify CRISPR’s basic architecture to extend its reach beyond the genome and into what’s known as the epigenome.
- CRISPR Therapies Pipeline Insights 2021: Analysis of Key Companies, Emerging Therapies, Recent Happenings and Futuristic Trendson April 12, 2021 at 5:31 pm
The leading gene-editing companies looking at commercializing CRISPR-based therapeutics are CRISPR Therapeutics, Intellia Therapeutics, and Editas Medicine. CRISPR Therapeutics has the largest market ...
- CRISPR Has a Problem: It Mangles DNA It Wasn’t Supposed to Touchon April 12, 2021 at 9:58 am
The gene-editing technology CRISPR Cas9 may not be the ultra-precise tool that researchers long thought it to be. A growing body of evidence suggests that the tech, which allows researchers to remove, ...
- Researchers call for greater awareness of unintended consequences of CRISPR gene editingon April 12, 2021 at 5:10 am
Researchers at the Francis Crick Institute have revealed that CRISPR-Cas9 genome editing can lead to unintended mutations at the targeted section of DNA in early human embryos. The work highlights the ...
- CRISPR Therapeutics Presents Preclinical Data at AACR 2021 Supporting CD70 Knockout as a Novel Approach to Increasing CAR-T Cell Functionon April 10, 2021 at 5:31 am
CRISPR Therapeutics (Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced preclinical data from the Company’s ...
- New, reversible CRISPR method can control gene expression while leaving underlying DNA sequence unchangedon April 9, 2021 at 8:00 am
Over the past decade, the CRISPR-Cas9 gene editing system has revolutionized genetic engineering, allowing scientists to make targeted changes to organisms' DNA. While the system could potentially be ...
via Bing News