Biology subjects

Terrace, C. I.

Publications and source records attributed to Terrace, C. I..

3 recordsLinked to original sources

bioRxiv2026

Activity and specificity trade-offs in adenine base editors

Adenine base editors (ABEs) are CRISPR effectors that introduce A-T to G-C transitions in the genome using a nucleotide deaminase fused to a Cas protein. ABEs have been evolved to have very high editing efficiency, but off-target editing effects compromise their precision and hinder their applications. Here we explore the activity and specificity relationship of ABEs using a combination of machine learning-guided design and high-throughput screening. We designed a diverse library of 12,000 variants and built quantitative bacterial selection systems that allowed us to simultaneously measure their on-target and off-target editing. We found that the ABEs were fully described by the single dimension of intrinsic deaminase activity with no evidence for independent specialization with respect to local sequence context, editing window width, RNA editing, or genotoxicity. These results were supported by in vitro studies and consistent with editing experiments in mammalian cells. Finally, the activity and specificity trade-offs were recapitulated among previously reported engineered variants and a selection of library variants spanning the activity spectrum. Our results suggest that fundamental architectural improvements will be necessary to transcend the activity and specificity limitations for the next generation of ABEs.

molecular biology↗

bioRxiv2025

Exploring the deletion landscape of S. aureus Cas9 with SABER

Profiling tolerated amino acid deletions in proteins can elucidate structure-function relationships, reconstruct intermediate stages in protein evolution, and be used to engineer minimized versions of proteins with size-sensitive biotechnology applications. Despite advances in deletion library construction techniques over the past several decades, there are presently few methods available that are simultaneously efficient, precise, and easy to implement. Here we present SABER, a novel approach which utilizes SpRYCas9, a near-PAMless engineered SpCas9 variant, as a molecular biological tool for building deletion libraries with unprecedented speed and ease. We applied this technique to the small and structurally divergent Cas9 from Staphylococcus aureus (SaCas9) and mapped the set of deletions tolerated for DNA binding activity. We proceeded to use this information to design a set of minimal SaCas9-based effectors capable of CRISPRi transcriptional repression in bacterial cells. Our findings provide new insights into the function of certain structural elements in SaCas9, and we anticipate that our dSaCas9 deletion map may prove useful in further efforts to develop minimal Cas9-based effectors and gene editors.

molecular biology↗

bioRxiv2025

Discovery of widespread activating mutations in a compact RNA-guided endonuclease

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the proteins mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

molecular biology↗