CRISPR-Cas9 Gene Editing: Check Three Times, Cut Once
By Robert Sanders | NOVEMBER 12, 2015
Two new studies from UC Berkeley should give scientists who use CRISPR-Cas9 for genome engineering greater confidence that they won’t inadvertently edit the wrong DNA.
The gene editing technique, created by UC Berkeley biochemist Jennifer Doudna and her colleague, Emmanuelle Charpentier, director of the Max Planck Institute of Infection Biology in Berlin, has taken the research and clinical communities by storm as an easy and cheap way to make precise changes in DNA in order to disable genes, correct genetic disorders or insert mutated genes into animals to create models of human disease.
The two new reports from Doudna’s lab and that of UC Berkeley colleague Robert Tjian show in much greater detail how the Cas9 protein searches through billions of base pairs in a cell to find the right DNA sequence, and how Cas9 determines whether to bind, or bind and cut, thereby initiating gene editing. Based on these experiments, Cas9 appears to have at least three ways of checking to make sure it finds the right target DNA before it takes the irrevocable step of making a cut.
“CRISPR-Cas9 has evolved for accurate DNA targeting, and we now understand the molecular basis for its seek-and-cleave activity, which helps limit off-target DNA editing,” said Doudna, a Howard Hughes Medical Institute investigator at UC Berkeley and professor of molecular and cell biology and of chemistry. Tjian is president of the Howard Hughes Medical Institute and a UC Berkeley professor of molecular and cell biology.
The studies also illustrate how well CRISPR/Cas9 works in human and animal cells – eukaryotes – even though “the technique was invented by bacteria to protect themselves from getting the flu,” Doudna said.
CRISPR-Cas9 is a hybrid of protein and RNA – the cousin to DNA – that functions as an efficient search-and-snip system in bacteria. It arose as a way to recognize and kill viruses, but Doudna and Charpentier realized that it could also work well in other cells, including humans, to facilitate genome editing. The Cas9 protein, obtained from the bacteria Streptococcus pyogenes, functions together with a “guide” RNA that targets a complementary 20-nucleotide stretch of DNA. Once the RNA identifies a sequence matching these nucleotides, Cas9 cuts the double-stranded DNA helix.
One study, published in the Nov. 13 issue of Science, tracked Cas9-RNA molecules though the nucleus of mammalian cells as they rapidly searched through the entire genome to find and bind just the region targeted and no other.
featured image: Jennifer Doudna, a professor of molecular and cell biology in the California Institute for Quantitative Biosciences (QB3) and an investigator in the Howard Hughes Medical Institute at Berkeley