The protospacer-adjacent motif specificity of several Cas enzymes is customized using protein language models.

A λ exonuclease system exhibits protospacer-adjacent motif-independent DNA-binding specificity, enabling diagnostic and imaging applications.

In this study, the authors track microbial communities in a perturbed cultivation vessel and show that methanogenic microbes develop resilience through CRISPR-Cas mutations and spacer expansions, driving phages to counter-adapt by altering infectivity genes and protospacer-adjacent motifs, revealing a host-phage arms race.

In this study, the authors track microbial communities in a perturbed cultivation vessel and show that methanogenic microbes develop resilience through CRISPR-Cas mutations and spacer expansions, driving phages to counter-adapt by altering infectivity genes and protospacer-adjacent motifs, revealing a host-phage arms race.

We developed Protein2PAM, an evolution-informed protein language model that predicts protospacer-adjacent motif (PAM) specificity of CRISPR–Cas enzymes. Using in silico mutagenesis and model-guided ev...

CRISPR–Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR–Cas12a and expanding the design space for programmable RNA manipulation. Synthetic DNA guides (crDNA) reprogram Cas12a nucleases for

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a fundamental defense mechanism in prokaryotes, where short sequences called spacers are stored in the host genome to rec...

CRISPR–Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, greatly limiting the range of targetable sequences in a genome. Although engineering strategies to alter PAM specificity exist, they typically require labor-intensive, iterative experimentation. We introduce an evolution-informed deep learning model, Protein2PAM, to efficiently guide the design of Cas protein variants tailored to recognize specific PAMs. Trained on a dataset of over 45,000 CRISPR–Cas PAMs, Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across type I, II and V CRISPR–Cas systems. Using in silico mutagenesis, the model identifies residues critical for PAM recognition in Cas9 without using structural information. We use Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild type in vitro. Our machine learning approach allows Ca

Background Asgard archaea, broadly considered as a group of archaea that exhibit the closest evolutionary relationship with eukaryotes, encode diverse defense systems and are infected by distinct viruses. However, the diversity of Asgard archaeal viruses and their interactions with the hosts remain poorly explored. Results Here, using CRISPR spacer-protospacer matching, we predict 23 viruses associated with Asgard archaea from various habitats. Apart from clusters with known viruses, we report three previously undescribed groups of Asgard viruses with predicted head-tailed virions of the realm Duplodnaviria and filamentous virions characteristic of the archaea-specific realm Adnaviria. Mapping of metagenomic reads to viral and host genomes suggested that most Asgard viruses display relatively low virus-to-host ratios, typically below 10. In addition, we considerably expanded the known defensome of Asgard archaea, among others, detecting putative PARIS and Prometheus defense systems. Finally, the diversity of anti-defense proteins and putative archaeal type IV pilins encoded by some Asgard viruses provide additional insights into the interactions between Asgard archaea and their viruses. Conclusions Overall, this work sheds new light on the Asgard archaeal virome and defensome, and virus-host interactions. Video Abstract

CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas