CRISPR Repairs DNA Even Without Donor Template
CRISPR-Cas9 can carry out precise genome editing even without the assistance of donor DNA templates, a team of scientists from Brigham and Women’s Hospital and the Broad Institute of MIT and Harvard have found. The research, published in the Nature journal, suggests that in the future the cell’s genetic auto-correction could be combined with CRISPR-based therapies to correct mutated genes by cutting the DNA precisely and allowing the cell to heal on its own.
In the past, CRISPR-Cas9 has been known for being “snip happy”, cutting a genomic site until a DNA repair system malfunctions and leaving the site scarred and less effective than precisely-edited DNA.
Many mutations associated with disease involve missing or extra DNA. Correcting these errors with CRISPR using a template of standard DNA is only effective in rapidly-dividing cells, and even then is not wholly effective. In order to restore gene function without templated repair, knowledge of how the cell will fix CRISPR-induced DNA breaks is a requirement, but until now this knowledge did not exist.
The new study created an algorithm to predict how human and mouse cells will respond to breaks in DNA created by CRISPR-Cas9, finding that cells will repair the damaged genes in predictable patterns and sometimes return them to full health. This research was taken further and used to correct cell mutations in patients with rare genetic disorders.
According to the scientists involved, a library of 2,000 Cas9 guide RNAs paired with DNA target sites was created, and a machine learning model was trained to accurately predict genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions in five human and mouse cell lines. The program: “…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 model, inDelphi, could identify patterns at genetic cut sites that predicted which insertions and deletions had been made in the fully-formed gene. Often the corrected gene needed only a single outcome to complete it, such as correction of a pathogenic gene. The scientists were able to correct nearly 200 such pathogenic genes in this manner, as well as mutations in cells from patients with Hermansky-Pudlak syndrome and Menkes disease.
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, said that: “…the same CRISPR enzyme that has been used primarily as a sledgehammer can also act as a chisel.
“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.”