Summary

Over billions of years, prokaryotes and their genetic parasites have coevolved, leading to the development of diverse defense systems in prokaryotic cells. In response, viruses and other mobile genetic elements have evolved strategies to counteract these defenses. Our research focuses on bacterial CRISPR-Cas adaptive immunity and phage-encoded anti-CRISPR mechanisms, with a special emphasis on type VI systems.

CRISPR-Cas systems consist of Clusters of Regularly Interspaced Short Palindromic Repeat (CRISPR) arrays and CRISPR-associated (Cas) proteins, functioning through three main stages. In the adaptation stage, DNA fragments from invading genetic elements are incorporated into the CRISPR array as spacers. Next, the CRISPR array is transcribed and processed into small CRISPR RNAs (crRNAs), each containing sequences from these spacers. Finally, during the interference stage, these crRNAs guide Cas proteins to the complementary regions in DNA or RNA targets, cleaving the latter and thereby protecting the host against phages and plasmids. The programmable Cas nucleases have revolutionized research and biomedicine. Among the evolutionarily and mechanistically diverse CRISPR-Cas systems, type VI systems stand out for their employment of single-subunit Cas13 effectors, which exclusively recognize and cleave RNAs. The RNA-targeting Cas13 nucleases have substantially expanded the arsenal of CRISPR technologies, being harnessed for powerful tools for transcription regulation and sensing, RNA modification and knockdown, and highly sensitive diagnostics.

Most CRISPR-Cas effectors guided by their crRNA to complementary target DNA destroy it, thus providing defense. In contrast, type VI Cas13 effectors cause a broad collateral effect. Upon target RNA binding, Cas13 RNase is activated, leading to target RNA cleavage and collateral cleavage of non-complementary RNAs (Figure 1). While target RNA primarily serves as an activator of Cas13 RNase, collateral RNA cleavage triggers a broad cellular response, inducing cell dormancy and effectively impeding the spread of invaders through the bacterial cell population. Our research aims to investigate the collateral RNA damage caused by different Cas13 homologs, their substrate preferences, and the defense pathways involved. We also explore potential anti-CRISPR mechanisms and how these systems acquire new spacers. We anticipate that our research will significantly enhance our understanding of the biological functions of CRISPR-Cas13 systems and drive the development of Cas13-based technologies.

The Mechanism Underlying the CRISPR-Cas13-Mediated Defense

We recently studied the collateral activity of the Cas13a effector from Leptotrichia shahii (LshCas13a) heterologously expressed in Escherichia coli cells. Using RNA-seq, we detected anticodon tRNA cleavage that did not depend on cellular RNases. In vitro cleavage assays with recombinant Cas13a confirmed that target-activated Cas13a is responsible for the tRNA cleavage detected in cells. We demonstrated that tRNA cleavage is sufficient for translation inhibition and suppression of cell growth during Cas13a interference, establishing it as the primary mechanism of antiphage defense provided by the type VI-A CRISPR-Cas system from L. shahii (Figure 2). Our serendipitous discovery that defense against phage infection conferred by LshCas13a involves tRNA cleavage introduces a new concept of collateral damage by Cas13 effectors, highlighting their evolutionary connection to HEPN-containing anticodon nucleases from abortive infection systems.

Defining the Specificity of Collateral Damage by Various Cas13 Homologs

Since the discovery of collateral RNA damage by target-activated Cas13, the role of this phenomenon in CRISPR immunity has been debated. It remains unclear whether different CRISPR-Cas13 systems predominantly rely on massive promiscuous RNA degradation or on more precise mechanisms like tRNA cleavage. Building on Cas13 collateral activity, highly sensitive nucleic acid detection tools have been developed. However, only a few Cas13 proteins have been found suitable for diagnostics due to the highly variable levels of collateral activity exhibited by different bacterial Cas13 proteins in vitro. Surprisingly, certain Cas13 effectors, when introduced into eukaryotic cells, demonstrate efficient target RNA knockdown with minimal or no collateral activity. These observations underscore the substantial functional variability within the Cas13 nuclease family and prompt questions about their substrate preferences for cleavage.

While investigating the collateral activity of LshCas13a, we developed an efficient pipeline for detecting RNA cleavage sites and analyzing the impact of collateral cleavage on cell metabolism and viability. We are currently using this pipeline to study various Cas13 homologs. In collaboration with Jennifer Doudna's lab (University of California, Berkeley), we are initially examining the specificity of collateral cleavage by Cas13 effectors from Leptotrichia buccalis (LbuCas13a), Listeria seeligeri (LseCas13a), and Ruminococcus flavefaciens (RfxCas13d). The LseCas13a system is well-characterized in native cells, providing a model for further study of Cas13 in its native context. RfxCas13d is the most commonly used Cas13 protein for RNA knockdown in eukaryotic cells, with multiple reports indicating a lack of collateral activity. LbuCas13a has been employed for diagnostics and as a counterselection tool in phage genome editing. Thus, accumulating data on these Cas13 systems will be valuable to align with our findings on cleavage preferences.

Our preliminary findings on RNA cleavages induced by LseCas13a upon RNA-targeting revealed interesting distinctions. Unlike LshCas13a, which exhibits specific cleavage of tRNAs, LseCas13a demonstrated a relaxed specificity, cleaving a broader range of RNAs, including mRNAs, rRNAs, and tRNAs. Notably, cleavage of tRNAs containing uridine-rich anticodons was detected in cells expressing LseCas13a upon RNA-targeting. These tRNAs are the preferred cleavage substrates for target-activated LshCas13a. The positions of tRNA cleavage at anticodon loops coincided for LseCas13a and LshCas13a. To further investigate LseCas13a cleavage specificity and defense mechanism, we intend to analyze whether translation is inhibited in the heterologous E. coli system expressing LseCas13a. Additionally, given the availability of the Listeria model system for Cas13 research, we are considering exploring LseCas13a cleavage in native cells by applying our RNA-seq protocol.

A recent study from Mitchell O'Connell’s lab (University of Rochester) revealed a CRISPR-Cas13b-assisted mechanism of antiphage defense through membrane pore activation. The accessory protein Csx28, encoded in the type VI-B2 CRISPR-Cas locus from Prevotella buccae (PbuCas13b), forms a membrane pore activated by PbuCas13b-mediated RNA cleavage. It has been suggested that PbuCas13b might cleave tRNAs, generating signals to activate the Csx28 pore. To investigate the specificity of PbuCas13b collateral cleavage, we initiated a collaboration with O'Connell's lab. Our preliminary experiments revealed RNA cleavage products whose sizes indeed suggest tRNA cleavage. We will further analyze PbuCas13b cleavage by RNA-seq. This research will complement structural and biochemical studies focused on Csx28, providing a comprehensive understanding of PbuCas13b's antiphage defense mechanism.

Exploration of Anti-CRISPR Mechanisms Subverting Cas13 Functions

In response to CRISPR-Cas immunity, phages and other genetic elements have evolved diverse counter-defense strategies. Our work focuses on unveiling anti-CRISPR mechanisms that could counteract the tRNA anticodon nuclease activity of Cas13. Our experiments with the T5 phage, which encodes numerous tRNAs, suggest that these tRNAs might help the phage evade destruction by CRISPR-Cas13 systems cleaving tRNAs. We are investigating whether phage tRNAs are resistant to cleavage by LshCas13a and exploring the potential role of tRNA repair systems in anti-CRISPR strategies.

Investigation of Spacer Acquisition by RNA-Targeting Type VI CRISPR-Cas Systems

We have developed an experimental model to study how type VI CRISPR-Cas systems acquire new spacers. For the first time, our work has revealed the functionality of the bona fide adaptation module of RNA-targeting type VI CRISPR-Cas systems. Our preliminary data show that double-stranded DNA can be incorporated into the CRISPR array, but RNA or RNA/DNA hybrids cannot. Notably, the Cas13 protein is not required for spacer acquisition, suggesting that the adaptation module operates independently. We plan to further explore the relationship between spacer acquisition and the interference activity of Cas13 proteins. Additionally, we intend to expand this experimental model to study spacer acquisition in other type VI CRISPR-Cas systems.