Medicilon Logo
search icon search icon language icon contact icon menu icon
Medicilon Logo
search icon close search icon language icon contact icon menu icon
Contact Us
Close Button
Back To Top
Online Message×
Click switch
Close Button
Medicilon's News information
News information

CRISPR-Cas9 without Donor Templates Still Accomplishes DNA Repair

Page View:

CRISPR-Cas9 has the reputation for being “snip happy,” for cutting and cutting a genomic site until one of the cell’s DNA repair systems finally skips a beat, leaving the site imperfectly repaired – scarred, really. Although scarred DNA has its purposes, it isn’t the same thing as precisely edited DNA.

According to a new study led by Brigham and Women’s Hospital and the Broad Institute of MIT and Harvard, CRISPR-Cas9 is capable precise genome editing – even without the assistance of donor DNA templates. CRISPR-Cas9’s previously unrecognized repair capabilities were first characterized in a machine learning model. Then they were confirmed in mouse and human cell models.

Detailed findings appeared in the journal Nature, in an article titled, “Predictable and precise template-free CRISPR editing of pathogenic variants.” Template-free Cas9 editing, the article maintains, is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. Essentially, the article suggests that the cell’s genetic auto-correction could one day be combined with CRISPR-based therapies that correct gene mutations by simply cutting DNA precisely and allowing the cell to naturally heal the damage.

When DNA is damaged in the absence of donor DNA, the usual DNA repair mechanism is an error-prone process called non-homology end joining (NHEJ). It is useful for disabling a gene, but researchers have deemed it too error-prone to exploit for therapeutic purposes.

The new study upends this view. By creating a machine-learning algorithm that predicts how human and mouse cells respond to CRISPR-induced breaks in DNA, the Brigham/Broad researchers discovered that cells often repair broken genes in ways that are precise and predictable, sometimes even returning mutated genes back to their healthy version. In addition, the researchers put this predictive power to the test and successfully corrected mutations in cells taken from patients with one of two rare genetic disorders.

“We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines,” the authors of the Nature article wrote. “inDelphi predicts that 5–11% of Cas9 guide RNAs targeting the human genome are ‘precise-50’, yielding a single genotype comprising greater than or equal to 50% of all major editing products.”

The researchers found that inDelphi could discern patterns at cut sites that predicted what insertions and deletions were made in the corrected gene. At many sites, the set of corrected genes did not contain a huge mixture of variations, but rather a single outcome, such as correction of a pathogenic gene.

Indeed, after querying inDelphi for disease-relevant genes that could be corrected by cutting in just the right place, the researchers found nearly two hundred pathogenic genetic variants that were mostly corrected to their normal, healthy versions after being cut with CRISPR-associated enzymes. They were also able to correct mutations in cells from patients with two rare genetic disorders, Hermansky-Pudlak syndrome and Menkes disease.

“We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky–Pudlak syndrome and Menkes disease,” the Nature article noted. “This study establishes an approach for precise, template-free genome editing.”

Many disease-associated mutations involve extra or missing DNA, known as insertions and deletions. Researchers have tried to correct those mutations with CRISPR-based gene editing. To do this, they cut the double helix with an enzyme and insert missing DNA, or remove extra DNA, using a template of genetic material that serves as a blueprint. The approach, however, only works in rapidly dividing cells like blood stem cells and even then it is only partly effective, making it a poor choice for therapeutics aimed at the majority of cell types in the body. To restore gene function without templated repair requires knowing how the cell will fix CRISPR-induced DNA breaks – knowledge that did not exist until now.

“We show that the same CRISPR enzyme that has been used primarily as a sledgehammer can also act as a chisel,” said Richard Sherwood, a corresponding author of the current study and an assistant professor of medicine in the division of genetics at Brigham and Women’s Hospital. “The ability to know the most likely outcome of your experiment before you do it will be a real advance for the many researchers using CRISPR.”

Relevant newsRelevant news