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Natural systems such as CRISPR-associated transposons (CASTs) offer a targetable, one-step way to edit genomes. However, adapting them for biomedical applications has been challenging.

To address this limitation, scientists at St. Jude Children's Research Hospital designed a screening approach to measure efficiency and specificity for thousands of CAST variants accurately.

This high-throughput approach allowed the researchers to rapidly optimize promising candidate CASTs, uncovering mechanistic insights fundamental for further engineering and potential clinical use.

The findings were in Nucleic Acid Research.

Discovered in 2017, CASTs integrate large pieces of DNA into the genome at locations defined by a given RNA sequence. They are highly specific in bacteria, their natural host, but they do not work as well in human cells.

For this reason, corresponding author Elizabeth Kellogg, Ph.D., St. Jude Department of Structural Biology, is keenly interested in engineering CASTs to be more applicable to humans and other organisms. To do so requires a quick way to evaluate the strengths and weaknesses of engineered CASTs.

"One major gap in understanding CASTs was not just measuring their overall activity, but also how accurately they integrate DNA at the intended locations," Kellogg said. "There simply wasn't a scalable way to characterize their specificity."

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, Ph.D., 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 five-fold," 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."