U.S. researchers improve CRISPR-Cas9 gene editing technique

Researchers at University of California, Berkeley, have improved CRISPR-Cas9 technology and achieved a success rate of 60 percent when replacing a short stretch of DNA with another.

The advance in CRISPR, short for clustered regularly interspaced short palindromic repeats, using the Cas9 protein is useful when trying to repair genetic mutations that cause hereditary diseases, such as sickle cell disease or severe combined immune deficiency.

The study at the university on the U.S. west coast about gene editing, a major biotech research area in recent years, is published in the Jan. 21 issue of the journal Nature Biotechnology.

"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," explained Jacob Corn, scientific director of the Innovative Genomics Initiative (IGI). "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."

The IGI at UC Berkeley focuses on next-generation genome editing and gene regulation for laboratory and clinical application.

Christopher Richardson, an IGI post doctoral researcher and first author of the study, 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.

In addition, Richardson discovered that while the Cas9 protein hangs onto three of the cut ends of the two strands of DNA, 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 therefore can add templates containing changes that alter existing sequences in the genome, for purposes such as correcting a disease-causing mutation.

Reasoning that bringing the substitute template directly to the site of the cut would improve the patching efficiency, Richardson constructed a piece of DNA that matches the free DNA end and carries the genetic sequence to be inserted at the other end. It worked well, allowing repair of a mutation with up to 60 percent efficiency.

The researchers hailed the success rate as "unprecedented."

"In cases where you want to change very small regions of DNA, up to 30 base pairs, this technique would be extremely effective," said Richardson.

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.

The CRISPR-Cas9 system allows researchers to patch an abnormal section of DNA with the normal sequence and potentially correct the defect. Endi