The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage

Liu, Shunhang; Rao, Xichen; Zhao, Ruiliang; Han, Wenyuan

doi:10.3389/fgeed.2022.929929

Cited by 6 publications

(4 citation statements)

References 22 publications

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“…To construct the pKD46-M1-EaAgo-EabAgaM plasmid, the p TR Cas12a-NT plasmid from our previous study 50 was used as template to amplify the Chl-resistant gene and the araBAD promoter-araC-pSC101 origin fragment. Then, the fragments were assembled with the fragment containing EaAgo and EabAgaM genes amplified from pBAD24-EaAgo-EabAgaM.…”

Section: Methodsmentioning

confidence: 99%

Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection

Song,

Lei,

Liu

et al. 2023

Nat Commun

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Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos) play potential functions in microbial defense. The functions and mechanisms of a group of full-length yet catalytically inactive pAgos, long-B pAgos, remain unclear. Here, we show that most long-B pAgos are functionally connected with distinct associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. The long-B pAgo-nuclease system (BPAN) is activated by guide RNA-directed target DNA recognition and performs collateral DNA degradation in vitro. In vivo, the system mediates genomic DNA degradation after sensing invading plasmid, which kills the infected cells and results in the depletion of the invader from the cell population. Together, the BPAN system provides immunoprotection via abortive infection. Our data also suggest that the defense strategy is employed by other long-B pAgos equipped with distinct associated proteins.

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Section: Methodsmentioning

confidence: 99%

Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection

Song,

Lei,

Liu

et al. 2023

Nat Commun

Self Cite

View full text Add to dashboard Cite

show abstract

“…To construct the pKD46-M1-EaAgo-EabAgaM plasmid , the p TR Cas12a-NT plasmid from our previous study 52 was used as template to amplify the Chl-resistant gene and the araBAD promoter-araC-pSC101 origin fragment. Then, the fragments were assembled with the fragment containing EaAgo and EabAgaM genes amplified from pBAD24-EaAgo-EabAgaM.…”

Section: Clonexpress II One Step Cloning Kit To Replace the Origin Of...mentioning

confidence: 99%

Long-B prokaryotic Argonaute systems employ various effectors to confer immunity via abortive infection

Song

Lei

Liu

et al. 2023

Preprint

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Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Prokaryotic Agos (pAgos) are extremely diverse, with potential functions in microbial defense. The functions and mechanisms of a group of full-length yet inactive pAgos, long-B pAgos, remain enigmatic. Here, we show that most long-B pAgos constitute cell suicide systems together with their various associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. Among them, the long-B pAgo-nuclease system utilizes an RNA-programmed and target-recognition-activated collateral DNA cleavage activity to sense invaders and kill the infected cells. This results in depletion of the invading plasmid from the cell population. Together, our data indicate that the long-B pAgo systems induce cell death with various effector proteins after recognition of invading nucleic acids, corresponding to an immune response via abortive infection.

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“…To date the available data suggest that Cas12a provides bacterial defense against mobile genetic elements (MGE) mainly through specific cis -cleavage of invading dsDNA, but not ssDNA targets ( 49 ), without any role played by non-specific trans -substrate cleavage ( 49 , 50 ). Among our findings are several that appear paradoxical to this mechanism, namely that ssDNA may activate Cas12a RNPs more rapidly than dsDNA targets do and that both targets activate RNPs to the same high level of catalytic activity for trans -cleavage, which is considerably higher against ssDNA than dsDNA trans -substrates ( 27 ).…”

Section: Discussionmentioning

confidence: 99%

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets

Nalefski,

Kooistra,

Parikh

et al. 2024

Nucleic Acids Research

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The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

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The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage

Cited by 6 publications

References 22 publications

Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection

Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection

Long-B prokaryotic Argonaute systems employ various effectors to confer immunity via abortive infection

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets

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