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.
“In a shorter time, I can target many genes,” Co-lead author Gaurav Varshney of the NHGRI told GenomeWeb. “Instead of doing 100 individual injections, I can do 10 injections. Instead of doing 100 out-crossings, I can do 10.”
And soon scientists could create triple mutants, targeting three potentially related genes at the same time. Doing such studies in mice is difficult because of the mathematics of animal husbandry, Burgess said, since only about one in 64 will have all three genes knocked out and mouse litters are only about eight to 12 pups. “Zebrafish can generate several hundred embryos, so you can get a number of [mutants] just by doing brute force genetics,” he said. “We’re moving in that direction now, and it’s actually working in our hands.”
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
SOURCES – Genome Web, Genome Research