TemStaPro: protein thermostability prediction using sequence representations from protein language models.

MOTIVATION: Reliable prediction of protein thermostability from its sequence is valuable for both academic and industrial research. This prediction problem can be tackled using machine learning and by taking advantage of the recent blossoming of deep learning methods for sequence analysis. These methods can facilitate training on more data and, possibly, enable the development of more versatile thermostability predictors for multiple ranges of temperatures. RESULTS: We applied the principle of transfer learning to predict protein thermostability using embeddings generated by protein language models (pLMs) from an input protein sequence. We used large pLMs that were pre-trained on hundreds of millions of known sequences. The embeddings from such models allowed us to efficiently train and validate a high-performing prediction method using over one million sequences that we collected from organisms with annotated growth temperatures. Our method, TemStaPro (Temperatures of Stability for Proteins), was used to predict thermostability of CRISPR-Cas Class II effector proteins (C2EPs). Predictions indicated sharp differences among groups of C2EPs in terms of thermostability and were largely in tune with previously published and our newly obtained experimental data. AVAILABILITY AND IMPLEMENTATION: TemStaPro software and the related data are freely available from https://github.com/ievapudz/TemStaPro and https://doi.org/10.5281/zenodo.7743637.

A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.