Biology subjects

Meeske, A. J.

Publications and source records attributed to Meeske, A. J..

7 recordsLinked to original sources

bioRxiv2026

Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems

Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri. Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.

microbiology↗

bioRxiv2025

Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data

CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model 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 deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ 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 under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.

synthetic biology↗

bioRxiv2024

Crosstalk between three CRISPR-Cas types enables primed type VI-A adaptation in Listeria seeligeri

CRISPR-Cas systems confer adaptive immunity to their prokaryotic hosts through the process of adaptation, where sequences are captured from foreign nucleic acids and integrated as spacers in the CRISPR array, and thereby enable crRNA-guided interference against new threats. While the Cas1-2 integrase is critical for adaptation, it is absent from many CRISPR-Cas loci, rendering the mechanism of spacer acquisition unclear for these systems. Here we show that the RNA-targeting type VI-A CRISPR system of Listeria seeligeri acquires spacers from DNA substrates through the action of a promiscuous Cas1-2 integrase encoded by a co-occurring type II-C system, in a transcription-independent manner. We further demonstrate that the type II-C integration complex is strongly stimulated by preexisting spacers in a third CRISPR system (type I-B) which imperfectly match phage targets and prime type VI-A adaptation. Altogether, our results reveal an unprecedented degree of communication among CRISPR-Cas loci encoded by a single organism.

microbiology↗

bioRxiv2024

Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences

Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology, and human health. CRISPR-based gene editors derived from microbes, while powerful, often show significant functional tradeoffs when ported into non-native environments, such as human cells. Artificial intelligence (AI) enabled design provides a powerful alternative with potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models (LLMs) trained on biological diversity at scale, we demonstrate the first successful precision editing of the human genome with a programmable gene editor designed with AI. To achieve this goal, we curated a dataset of over one million CRISPR operons through systematic mining of 26 terabases of assembled genomes and meta-genomes. We demonstrate the capacity of our models by generating 4.8x the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate an AI-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 publicly to facilitate broad, ethical usage across research and commercial applications.

synthetic biology↗

bioRxiv2023

Diverse viral cas genes antagonize CRISPR immunity

Prokaryotic CRISPR-Cas immunity is subverted via anti-CRISPRs (Acrs), small proteins that inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids. CRISPR-Cas defenses are classified into 6 types and 33 subtypes, which employ a diverse suite of Cas effectors and differ in their mechanisms of interference. As Acrs often work via binding to a cognate Cas protein, inhibition is almost always limited to a single CRISPR type. Furthermore, while acr genes are frequently organized together in phage-associated gene clusters, how such inhibitors initially evolve has remained unclear. Here we have investigated the Acr content and inhibition specificity of a collection of Listeria isolates, which naturally harbor four diverse CRISPR-Cas systems (types I-B, II-A, II-C, and VI-A). We observed widespread antagonism of CRISPR, which we traced to 12 novel and 4 known Acr gene families encoded on endogenous mobile genetic elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins and assemble into a defective interference complex. Surprisingly, an additional type I-B Cas homolog did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system through sequestration of crRNA. By probing the IMGVR database of viral genomes, we detected abundant orphan cas genes located within putative anti-defense gene clusters. We experimentally verified the Acr activity of one viral cas gene, a particularly broad-spectrum cas3 homolog that inhibits type I-B, II-A, and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution via cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.

microbiology↗

bioRxiv2022

Phage genome cleavage enables resuscitation from Cas13-induced bacterial dormancy

CRISPR-Cas systems provide their prokaryotic hosts with sequence-specific immunity to foreign genetic elements, including bacteriophages and plasmids. While most interfere with phage infection though cleavage of viral DNA, type VI CRISPR systems use the RNA-guided nuclease Cas13 to recognize mRNA targets. Upon engaging with target RNA, Cas13 cleaves both phage and host transcripts nonspecifically, leading to a state of cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is not clear. Here we show that type VI CRISPR systems frequently co-occur with DNA-cleaving restriction modification (RM) systems. Using genetics and microscopy, we show that Cas13 and RM systems synergize in anti-phage defense in the natural type VI CRISPR host Listeria seeligeri. Cleavage of the phage genome by RM removes the source of phage transcripts, enabling cells to recover from Cas13-induced cellular dormancy. We find that Cas13 and RM systems operating simultaneously eliminate phage DNA and neutralize infection more effectively than either defense alone. Thus, cells harboring both defense systems exhibit robust anti-phage immunity and survive infection. Our work therefore reveals that type VI CRISPR immunity is cell-autonomous and non-abortive, if paired with RM or similar DNA-targeting defenses. The ability of an abortive response to be resolved by the actions of another anti-phage defense has implications for the roles of diverse host-directed immune systems in bacteria.

microbiology↗

bioRxiv2021

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.

molecular biology↗