Researchers at the Wake Forest Institute for Regenerative Medicine (WFIRM) who are working on CRISPR/Cas9-mediated gene-editing technology have developed a technique to boost editing performance by reducing DNA deletion sizes, which is an important step toward developing gene-editing therapies to cure genetic diseases.
CRISPR (clustered regularly interspaced short palindromic repeats) technology is used to manipulate gene function and change DNA sequences. CRISPR/Cas9 is a DNA-editing enzyme that works by cutting two strands of DNA at a specified spot to add, delete, or repair fragments of DNA. Researchers seek to treat genetic illnesses by preventing a damaged cell from duplicating the flawed DNA by altering gene function.
CRISPR/Cas9 is the most powerful genetic alteration tool available, with numerous uses. While CRISPR/Cas9 is best known for producing small insertions or deletions at the target site, it can also produce massive DNA deletions around the target site. These extensive deletions raise safety concerns and may reduce the efficiency of functional editing. The WFIRM team is investigating strategies to lessen the likelihood of this happening.
According to lead author Baisong Lu, PhD, of WFIRM, the research presented in their latest publication, published recently by Nucleic Acids Research, intended to address the formation of unforeseen on-target lengthy DNA deletions and identify a means to guard against them.
Considering that a lack of methods to avoid generating long deletions by CRISPR/Cas9 is a challenge to the field, we are developing new counter-strategies. We developed a method to refine the mutations—to increase small deletions and decrease large deletions.”
Baisong Lu, PhD, Study Lead Author, Wake Forest Institute for Regenerative Medicine
The researchers looked at a variety of human cells and genes and discovered that connecting DNA polymerase I or the Klenow fragment to the Cas9 enzyme reduced the number of significant genomic DNA deletions while maintaining genome editing efficiency. In fact, in human basic cells, doing so boosted editing performance.
This technique increased genome editing efficiency in primary cells and did not increase DNA substitution rates or off-target rates. It also decreased large deletions, thus increasing safety. We can increase the percentage of desirable types of mutations. This improves the efficiency of disrupting disease-causing genes or restoring disrupted genes.”
Anthony Atala, MD, Study Co-Author and Director, Wake Forest Institute for Regenerative Medicine
Yoo, K. W., et al. (2022) Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9. Nucleic Acids Research. doi.org/10.1093/nar/gkac186.