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Multiplex Genome Engineering Platform Offers Biomanufacturing, Research Applications


NEW YORK (GenomeWeb) – A new multiplex genome editing system developed by researchers at the University of Colorado Boulder and Muse Biotechnology could accelerate biotechnology-based manufacturing.

CRISPR/Cas9-enabled trackable genome engineering (CREATE) enables researchers to make multiple kinds of mutations at multiple sites in a genome, CU Boulder researcher Ryan Gill told GenomeWeb. Led by Gill, the CREATE team published a paper describing their system Monday in Nature Biotechnology.

"In the past, people have designed multiplex guide RNAs [making one mutation type], or they've done single-guide RNAs with a multiplex repair cassette," he said. "We're the first to put those into a single oligonucleotide that allowed us to do multiple mutations at multiple loci."

The researchers combined several elements into a genome engineering package, including a barcoding strategy, high recombination efficiencies in both yeast and Escherichia coli, and multiplexing.

CREATE "shatters" the limitations in the "build" phase of the design-build-test cycle, Gill said, with implications not only for bio-fabrication but also for clinical research.

Gill's work on CREATE dates back before the advent of CRISPR as a genome editing tool. He's been working in the genome engineering field since the early 2000s, but quickly jumped on the CRISPR bandwagon. "The endonuclease selection before CRISPR worked perfectly for what we wanted to do, but CRISPR gave us what we needed to complete the technology package," he said.

While the ability to read genomes using sequencing has provided a much better picture of what might be important in the genomic landscape, he said that the ability to write genomes will accelerate the field even more quickly.

In the paper, the researchers used CREATE to reconstruct a prior experiment that used adaptive laboratory evolution (ALE) to study E. coli heat tolerance. In 2012, scientists from the University of California, Irvine published a study in Science describing almost a thousand mutations in E. coli that had adapted to a higher temperature. While sequencing offers the ability to infer what's important, recreating each mutation offers a direct route to finding the most important changes. Historically, in ALE and similar experiments, like directed evolution, fewer than 1 percent of all mutations end up being positive hits.

Gill and his co-authors wrote that they found all the mutations identified by the UC-Irvine team as well as 141 more. "There's really no other way to do that, to reconstruct all the mutations that might come out of an ALE study with deep sequencing," Gill said.

The ability to directly test the effects of a mutation isn't trivial, he said.

"At the pathway level, the ability to create 100,000 rational variants that you can then screen for production, that's a couple orders of magnitude greater than you might commonly find today," Gill said. "If you want to make a small molecule pharmaceutical, a recombinant protein, an amino acid, or a bunch of products we make using microbial systems, they typically require you to engineer 50 genes." Doing so takes a long time and a lot of money.

"Effectively, we're not limited anymore in the build step," he said, the traditional bottleneck. "Now the rate-limiting step is testing. Can we test 10,000 strains? Can we test 100,000 mutations? That's a good place to be."

While many of the applications Gill envisions will take place in cellular factories like yeast and E. coli, he sees potential in extending the idea into more complex eukaryotes.

The authors noted that CREATE in its current form is not equipped to deal with the non-homologous end joining repair pathway found in, say, mammalian cells, and that the barcoding systems — in this case the plasmids themselves — will need to be stably integrated. But Gill said there were some parallels between the numerous mutations found by sequencing in ALE studies and those found in clinical sequencing studies. CREATE in human cells would allow researchers to specifically test each mutation's effect on, say, cell growth.

The authors have created a publicly available CREATE cassette design tool for E. coli, available online. This tool features automated identification and synonymous mutation design of protospacer-adjacent motif sequences. Scaled for genome-wide engineering, the authors said they could design more than 100,000 cassettes using just an ordinary laptop.