Using CRISPR-CAS9 gene editing six times more effective for creating mutations

Scientists have developed a method for generating large numbers of mutated genes with CRISPR/Cas9 and a pipeline for identifying interesting mutations that enables high-throughput, multiplexed gene editing research.

"What this paper did was lay out a pipeline for making mutants at a relatively high throughput," senior author Shawn Burgess of the Translational and Functional Genomics Branch of the National Human Genome Research Institute told GenomeWeb. "Almost anything can be done on a small scale, but everything changes when you try to increase numbers."

The researchers were able to target zebrafish genes using CRISPR/Cas9, achieving germline mutations in about a quarter of the fish in the study. They targeted 162 locations in 83 genes, successfully mutating 82 of the 83 genes in the study, while using only about 1,000 fish. Burgess added that scientists in his lab have created about five times as many mutations in zebrafish with CRISPR/Cas9, but just haven't published data on them yet.

Creating mutations with CRISPR/Cas9 was six times more effective than other gene editing technologies, including both zinc finger nucleases and transcription activator-like effector nucleases, Burgess said, even when those other methods were optimized. And when comparing CRISPR/Cas9 to pre-gene editing technologies, like random mutagenesis followed by exome re-sequencing, it's not even close.

"You would have to go through usually five to ten thousand genomes to find the mutant you wanted," Burgess said. To find just one mutation, the cost of reagents alone could be $20,000 to $30,000. "Now the reagent cost for this is $30," he said.

Multiplex gene editing had been demonstrated in mouse and zebrafish before, Burgess said, but this paper showed it was possible to do it in a large-scale, repetitious fashion.