Advance improves cutting and pasting with CRISPR-Cas9 gene editing
University of California, Berkeley
January 20, 2016
Berkeley researchers have made a major improvement in CRISPR-Cas9 technology that achieves an unprecedented success rate of 60 percent when replacing a short stretch of DNA with another.
|Researchers Improves CRISPR's Cut-and-Paste Functionality|
“The exciting thing about CRISPR-Cas9 is the promise of fixing genes in place in our genome, but the efficiency for that can be very low,” said Jacob Corn, scientific director of the Innovative Genomics Initiative at UC Berkeley, a group that focuses on next-generation genome editing and gene regulation for lab and clinical application. “If you think of gene editing as a word processor, we know how to cut, but we need a more efficient way to paste and glue a new piece of DNA where we make the cut.”
“In cases where you want to change very small regions of DNA, up to 30 base pairs, this technique would be extremely effective,” said first author Christopher Richardson, an IGI postdoc.
Problems in short sections of DNA, including single base-pair mutations, are typical of many genetic diseases. Base pairs are the individual building blocks of DNA, strung end-to-end in a strand that coils around a complementary strand to make the well-known helical, double-stranded DNA molecule.
Richardson, Corn and their IGI colleagues describe the new technique in the Jan. 21 issue of the journal Nature Biotechnology.
Grabbing onto a loose strand
Richardson invented the new approach after finding that the Cas9 protein, which does the actual DNA cutting, remains attached to the chromosome for up to six hours, long after it has sliced through the double-stranded DNA. Richardson looked closely at the Cas9 protein bound to the two strands of DNA and discovered that while the protein hangs onto three of the cut ends, one of the ends remains free.
When Cas9 cuts DNA, repair systems in the cell can grab a piece of complementary DNA, called a template, to repair the cut. Researchers can add templates containing changes that alter existing sequences in the genome — for example, correcting a disease-causing mutation.
Richardson reasoned that bringing the substitute template directly to the site of the cut would improve the patching efficiency, and constructed a piece of DNA that matches the free DNA end and carries the genetic sequence to be inserted at the other end. The technique worked extremely well, allowing successful repair of a mutation with up to 60 percent efficiency.
The researchers also showed that variants of the Cas9 protein that bind DNA but donot cut also can successfully paste a new DNA sequence at the binding site, possibly by forming a “bubble” structure on the target DNA that also acts to attract the repair template. Gene editing using Cas9 without genome cutting could be safer than typical gene editing by removing the danger of off-target cutting in the genome, Corn said.
Co-authors with Richardson and Corn are IGI researchers Jordan Ray, Mark DeWitt and Gemma Curie. The work was funded by the Li Ka Shing Foundation.
News Release Source : Advance improves cutting and pasting with CRISPR-Cas9 gene editing
Image Credit : University of California, Berkeley