Tiny protein offers new path for gene editing

Good news! Will this discovery amend or enhance CRISPR-Cas (discovered in 2012)?

"Scientists have used protein engineering and an AI model to make the bacterial protein TnpB an effective gene editing tool for mammalian cells. TnpB is much smaller than the CRISPR-Cas system and could be more easily delivered to the right cells of the body as a result.

“By engineering the small but powerful protein TnpB, we were able to design a variant that shows a 4.4-fold increase in efficiency of modifying DNA – making it more effective as a gene editing tool,” ..."

"... It was recently discovered that Cas proteins evolved from much smaller proteins, with TnpB being the progenitor of Cas12. Since the large size of Cas proteins creates challenges when trying to deliver them to the right cells in the body, recent studies tried to use their smaller evolutionary progenitors as a genome editing tool. The problem with these small alternatives is that they function less efficiently. ...

TnpB proteins are found in a variety of bacteria and archaea. The TnpB studied by the researchers comes from the bacterium Deinococcus radiodurans. This microbe survives cold, dehydration, vacuum and acid, and is one of the most radiation-resistant organisms known to humans. The compact TnpB protein has previously been shown to work for genome editing in human cells, albeit with low efficiency and limited targeting ability due to its recognition requirements when binding DNA. ...

Therefore, the researchers optimized TnpB so that it edits the DNA of mammalian cells more efficiently than the original protein. “The trick was to modify the tool in two ways: first, so that it more efficiently goes to the nucleus where the genomic DNA is located, and second, so that it also targets alternative genome sequences,” ..."

From the abstract:

"Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified thus far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3% in the murine liver and 65.9% in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics."

Tiny protein offers new path for gene editing

Compact “Gene Scissor” Enables Effective Genome Editing (original news release) "CRISPR-Cas is used broadly in research and medicine to edit, insert, delete or regulate genes in organisms. TnpB is an ancestor of this well-known “gene scissor” but is much smaller and thus easier to transport into cells. Using protein engineering and AI algorithms, UZH researchers have now enhanced TnpB capabilities to make DNA editing more efficient and versatile, paving the way for treating a genetic defect for high cholesterol in the future."

Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs (no public access)