Antiviral signalling by a cyclic nucleotide activated CRISPR protease

Abstract

CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNAtargeting type III systems are widespread in prokaryotes 1,2 . The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5 . Among a large set of proteins that were linked to type III systems and predicted to bind cOAs 6,7 , a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODSassociated and fused to various effector domains) family 7 , fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazFhomolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS).3 Main CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptative immune system that enables microorganisms to fend off attacks from mobile genetic elements such as phages, viruses, or plasmids 8 . The protein complex Cas1-Cas2 captures short DNAs from invaders and integrates them as "memories" into a CRISPR locus 9 . Transcripts of these "memories" are processed into small CRISPR RNAs (crRNAs) and integrated into large ribonucleoprotein (RNP) complexes, which can sense the presence of a matching foreign nucleic acid in the cell 10 . Once an invading sequence is detected, an antiviral response is triggered. Depending on the type of CRISPR system 1 , this response can be markedly different, ranging from cleavage of the invading nucleic acid by the RNP as in the case of Cas9 11 , to a complex multipronged defense strategy as found in type III CRISPR systems 12 . For the latter, the Cas10 subunit of the RNP has a cyclase activity that converts ATP into a recently discovered class of cyclic oligoadenylates (cOAs) upon viral RNA recognition [3][4][5] . The cOAs are constructed from 3 to 6, 3'-5' linked AMP units 13 and act as second messengers, typically by binding to proteins harboring a CARF (CRISPR-associated Rossmann-fold) domain 14 . There is a wide variety of CARF proteins linked to effector domains with functions ranging from RNA cleavage, supercoiled DNA nicking, dsDNA cleavage to transcription modulation 12,[15][16][17][18][19][20] . The downstream effects of those cOA-activated proteins can lead to viral clearance, an abortive infection or a dormant state of the cell, enabling it to weather the phage attack 18,21 .Recently, two bioinformatic teams cataloged C...

CRISPR-based nucleic acid diagnostics for pathogens

“…Strategies using tandem CRISPR nucleases, and CRISPR with other catalytic molecules (such as enzymes and DNAzymes) have been reported to substitute nucleic acid amplification, and are promising to construct preamplification-free assays. Particularly, the recently reported Craspase [ 32 ], CalpL [ 35 ] and TIR-SAVED [ 36 ], the RNA-activated CRISPR protease or NADase that differs from conventional Cas effectors with nuclease function, has the potential for amplified detection via using protein reporters with high catalytic activities. Second, the improvement of assaying specificity capable of single-nucleotide discrimination is critical for diagnosis of pathogenic variants and mutations.…”

“… CRISPR tool Pathogens Analytes Preamplification strategy Assaying time Sensitivity Samples Ref. Type I CRISPR/Cas3 Virus SARS-CoV-2 RT-LAMP 30–40 min 10 2 copies Nasopharyngeal and oropharyngeal swab [ 21 ] Type III-A CRISPR/Cas assisted with Can2 Virus SARS-CoV-2 – 35 min 90 fM Nasopharyngeal swab [ 35 ] Type III-A CRISPR/Cas assisted with Csm6 Virus SARS-CoV-2 RT-LAMP 30 min 200 copies/μL Nasal swab [ 38 ] Type III-A CRISPR/Cas assisted with Csm6 Virus SARS-CoV-2 RT-RPA 30 min 60 copies/μL Nasopharyngeal swab [ 39 ] Cas13a assisted with Csm6 Virus SARS-CoV-2 – 20 min 30 copies/μL Respiratory swab [ 67 ] Cas9 Virus ZIKV NASBA 2–6 h 2.8 fM Plasma [ 83 ] Cas9 Virus SARS-CoV-2 VOCs RT-PCR 1.5 h A few copies Clinical samples [ 99 ] Cas9 Bacteria Salmonella LAMP 30 min 100 copies – [ 117 ] Cas9 Bacteria MRSA PCR …”

“…The RNA-activated protein cleavage using Craspase can be explored to sense RNAs either in vitro or inside cells [ 34 ]. In particular, a CRISPR protease (CalpL) [ 35 ] and a NADase (TIR-SAVED) [ 36 ] that both can be activated by cyclic nucleotides (cA 4 or cA 3 ) are recently identified. Combining the proteinase and NADase effectors with CRISPR type III systems holds great potential to develop sensitive assays for RNA sensing.…”

Everything OLD is new again: How structural, functional, and bioinformatic advances have redefined a neglected nuclease family

“…anti-phage signaling system), and PYCSAR (pyrimidine cyclase system for anti-phage resistance) use cyclic nucleotides as second messengers to activate a diverse range of effector proteins that in turn provide the critical defense activities (Athukoralage & White, 2022;Cohen et al, 2019;Duncan-Lowey & Kranzusch, 2022;Govande et al, 2021;Kazlauskiene et al, 2017;Lau et al, 2020;Manik et al, 2022;Millman, Melamed, et al, 2020;Niewoehner et al, 2017;Ofir et al, 2021;Rouillon et al, 2018;Rouillon et al, 2023;Tal et al, 2021). The Wadjet system recognizes DNA topology in closed circular DNA to protect hosts against plasmid transformation (Deep et al, 2022).…”

“…CRISPR‐Cas systems function as an adaptive immune system, able to acquire short segments of viral DNA from previous infections and archiving them into arrays with defined spacer sequences that guide the associated interference machinery to target and destroy complementary sequences upon subsequent infections (Hille et al, 2018; Watson et al, 2021), while abortive infection systems like AbiA, AbiK, Lit, and anti‐codon nucleases like PrrC and RloC behave as suicide switches that induce cell death as an antiviral defense (Blanga‐Kanfi et al, 2006; Davidov & Kaufmann, 2008; Kao & Snyder, 1988; Tangney & Fitzgerald, 2002; Wang et al, 2011). Some systems employ chemical modulators to disrupt phage replication directly (Kronheim et al, 2018) while others like Type III CRISPR complexes, Thoeris, CBASS (cyclic nucleotide‐based anti‐phage signaling system), and PYCSAR (pyrimidine cyclase system for anti‐phage resistance) use cyclic nucleotides as second messengers to activate a diverse range of effector proteins that in turn provide the critical defense activities (Athukoralage & White, 2022; Cohen et al, 2019; Duncan‐Lowey & Kranzusch, 2022; Govande et al, 2021; Kazlauskiene et al, 2017; Lau et al, 2020; Manik et al, 2022; Millman, Melamed, et al, 2020; Niewoehner et al, 2017; Ofir et al, 2021; Rouillon et al, 2018; Rouillon et al, 2023; Tal et al, 2021). The Wadjet system recognizes DNA topology in closed circular DNA to protect hosts against plasmid transformation (Deep et al, 2022).…”