Researchers discover new class of guide RNA for genome editing

A team of researchers has used cut-and-paste mobile genetic elements (MGEs) from the insert sequence (IS)110 family and clues from noncoding (nc)RNA to determine that large-scale genome design could be a possibility through a new class of guide RNAs.

The potential breakthrough came from asking whether ncRNA might assist recombinase in recognizing the target DNA site or the donor DNA (that is, the IS110 element itself), according to Drs. Matthew Durrant and Nicholas Perry of the Arc Institute in Palo Alto, CA. Together, Durrant, a computational biologist and senior scientist at Arc, and Perry, a PhD graduate student at the University of California (UC), Berkeley, led the experimental study with Dr. Patrick Hsu at Arc's Patrick Hsu lab.

Aided by cryo-electron microscopy analysis and nanopore sequencing, the study used Escherichia coli (E. coli) for its large, circular molecule of DNA chromosome and small, circular molecule plasmids.

In June, researchers confirmed a mechanism for a programmable target loop that allows the user to specify any desired genomic target sequence and any donor DNA molecule to be inserted. The development, detailed in the journal Nature, could eventually lead to a new genome editing method that sidesteps CRISPR DNA-cutting techniques, according to Arc.

The key, researchers discovered, lies in a new class of guide RNA, called "bridge RNA," that connects target and donor DNA and enables recombination by the IS621 recombinase. IS621, which resides in the IS110 family and is native to some strains of E. coli, as well as five closely related orthologues, was a central focus of this research, according to Durrant and colleagues.

"The bridge RNA system is a fundamentally new mechanism for genome design," said Hsu, senior author of the study and an Arc Institute core investigator and UC Berkeley assistant professor of bioengineering, in "Genomes by Design," an Arc blog post. "Bridge recombination can universally modify genetic material through sequence-specific insertion, excision, inversion, and more, enabling a word processor for the living genome beyond CRISPR."

Arc describes the discovery as a compact and entirely new type of programmable molecular system.

First, the team constructed a custom sequence database of bacterial isolate and metagenomic sequences by aggregating publicly available sequence databases.

As explained in Nature, the work investigated the potential presence of an IS110-encoded ncRNA by focusing on IS621. Researchers also evaluated the ncRNA consensus secondary structure across 103 diverse orthologues.

Durrant and colleagues found that ncRNA is necessary for in vitro recombination, and that the four components (ncRNA, recombinase, target DNA, and donor DNA) are sufficient to produce the expected recombination product. In addition, the base-pairing mechanism of target and donor recognition by the bridge RNA suggested programmability.

To assess programmability, the team designed an E. coli selection screen linking thousands of barcoded pairs of DNA targets and bridge RNAs on a single plasmid. This step helped to assess mismatch tolerance and reprogramming rules of bridge RNAs. They reprogrammed bridge RNAs to target sequences found only once in the E. coli genome.

"Altogether, these experiments provide evidence of the robust capability of IS621 to specifically insert multi-kilobase cargos into the genome, and offer further insights into the mechanisms of recombination," Durrant and colleagues wrote.

"The system can go far beyond its natural role that inserts the IS110 element itself, instead enabling insertion of any desirable genetic cargo — like a functional copy of a faulty, disease-causing gene — into any genomic location," Arc explained, adding that Hsu and colleagues demonstrated over 60% insertion efficiency of a desired gene in E. coli with over 94% specificity for the correct genomic location.

According to Arc, the Hsu lab found that when IS110 excises itself from a genome, the non-coding DNA ends are joined together to produce an RNA molecule — the bridge RNA — that folds into two loops. One loop binds to the IS110 element itself, while the other loop binds to the target DNA where the element will be inserted.

"We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules," the researchers explained in Nature. "The bridge RNA that we discovered in this work is the first example, to our knowledge, of a bispecific guide molecule that encodes modular regions of specificity for both the target and the donor DNA, coordinating these two DNA sequences in close proximity to catalyse efficient recombination."

Arc Institute operates in collaboration with Stanford University, UC Berkeley, and the University of California, San Francisco, according to information on the institute's website. The bridge RNA study included collaborators Hiroshi Nishimasu and Masahiro Hiraizumi at the University of Tokyo.

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