“Move over CRISPR, the retrons are coming!” Research scientists from the Wyss Institute for Biologically Inspired Engineering, Harvard University and Harvard Medical School (HMS) have come up with a new first of its kind gene-editing tool called Retron Library Recombineering (RLR) that enables millions of genetic experiments to be performed simultaneously.
This element uses single-stranded DNA for recombineering instead of depending on CRISPR-Cas–directed genomic breaks for genome editing. It now enables the construction of millions of libraries and allows natural DNA or random variation to be used as inputs.
Retrons vs CRISPR
Difficult to deliver in larger numbers.
It can be harmful to cells.
For a long time, CRISPR was just considered a weird thing that bacteria did, and figuring out how to harness it for genome engineering changed the world. They are another bacterial innovation that might also provide some important advances. – MAX SCHUBERT
Here, the targeted reverse-transcription activity of retrons was used to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. Retron Library Recombineering simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
“Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other,” said senior author George Church, who leads the Wyss Institute’s Synthetic Biology Focus Area.
Max G. Schubert et. al., High-throughput functional variant screens via in vivo production of single-stranded DNA. PNAS May 4, 2021, 118 (18).
New Gene-Editing Tool- Wyss Institute News