CASTing call for specificity and efficiency
To address this gap, the researchers devised a high-throughput screen to measure the relative activity and specificity of thousands of CAST variants in a single experiment. By focusing on a single subtype — the V-K CAST, which is notably less complex than others — they were able to make slight alterations to its proteins and rapidly screen the mutants.
“We wanted to screen a library with every possible single mutation to explore the mutational landscape of a CAST system,” said co-first author Seong Guk Park, PhD, Department of Structural Biology. “We didn’t focus on specific regions; instead, we tested them all to find mutations that could improve activity or specificity.”
After the V-K CAST mutational screening, the researchers combined several of the most promising mutations to see if their positive effect was additive. “With just a few mutations, activity increased fivefold,” Kellogg said. “We improved both specificity and activity without compromising either, which was not possible with previous strategies.”
Kellogg will continue working to improve the CAST design. With this screen now available, she is optimistic. “The natural system is very complicated, so we need to develop more minimal systems,” Kellogg said. “But this screen now enables us to be more ambitious with our protein design so that we can ultimately achieve this goal.”
Reference: Park SG, Park JU, Dodero-Rojas E, Bryant JA, Sankaranarayanan G, Kellogg EH. Comprehensive profiling of activity and specificity of RNA-guided transposons reveals opportunities to engineer improved variants. Nucleic Acids Res. 2025;53(18):gkaf917. doi: 10.1093/nar/gkaf917
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