A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13

Vialetto, Elena; Yu, Yanying; Collins, Scott P.; Wandera, Katharina G; Barquist, Lars; Beisel, Chase L.

doi:10.1016/j.chom.2022.05.013

Cited by 14 publications

(7 citation statements)

References 76 publications

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“…In both cases, we found that resolving predicted secondary structures that would interfere with gRNA function restored antimicrobial activity. Such an impact on gRNA folding has been observed previously for different CRISPR-Cas systems ( 56 , 59 , 60 , 62 , 69–71 ). However, the connection between gRNA folding and strain-specific antimicrobial activity is likely dependent on other factors.…”

Section: Discussionsupporting

confidence: 77%

“…As part of these experiments, SpCas9 and AsCas12a displayed strong antimicrobial activity, with a respective ∼10 3 –10 4 -fold and ∼10 4 –10 5 -fold reduction in transformants compared to a non-targeting gRNA control (Figure 1B ). In contrast, Cas13a exhibited negligible antimicrobial activity for all targets, possibly due to insufficient nuclease or target expression as we reported previously ( 56 ). Finally, SuCas12a2 and MpCas12a2 exhibited a strong reduction in colony numbers for the two essential gene targets (>10 4 -fold and >10 3 -fold, respectively) but a negligible reduction for the two non-essential gene targets.…”

Section: Resultssupporting

confidence: 63%

“…promoter strength, target site selection), as previous studies have reported potent antimicrobial activity with this nuclease type in other bacteria ( 21 ). Another possible explanation is that the target transcripts did not exceed a minimal expression threshold to enact cell dormancy ( 56 ), which could limit possible targets for Cas13-based antimicrobials. The lack of colony reduction when targeting non-essential genes with Cas12a2 was surprising given its ability to enact collateral cleavage of dsDNA that shuts down the cell regardless of the original target location ( 54 , 55 ).…”

Section: Discussionmentioning

confidence: 99%

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Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity

Vialetto,

Miele,

Goren

et al. 2024

Nucleic Acids Research

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CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.

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Section: Discussionsupporting

confidence: 77%

Section: Resultssupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

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Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity

Vialetto,

Miele,

Goren

et al. 2024

Nucleic Acids Research

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“…1b, c ). To explicitly evaluate the impact of expression strength, we replaced the strong constitutive promoter in front of the lacZ1 - and lolD -targeting sgRNAs with two weaker versions: a medium promoter (J23116) and a weak promoter (J23109) 20 . The weak promoter yielded similarly high colony counts (~10 5 − 10 6 transformants) for WT and Δ recA cells for both sites, indicative of cell survival.…”

Section: Resultsmentioning

confidence: 99%

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Collias

Vialetto

et al. 2023

Nat Commun

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Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

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“…Other reports of collateral cleavage or toxicity by Cas13 have also observed that these effects are target RNA expression level dependent[51,52]. Even in bacteria, there seems to be an expression threshold which must be reached to induce dormancy or cell death[66]. This suggests that a single target RNA activates only one or at most a few Cas13 proteins, probably due to prolonged guide RNA-target RNA base pairing after activation.…”

Section: Discussionmentioning

confidence: 98%

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

Bot

Zhao

Kammeron

et al. 2023

Preprint

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CRISPR-Cas13 systems are unique among Class II CRISPR systems, as they exclusively target RNA. In vitro and in prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA. This property has been exploited for development of diagnostic nucleic acid detection tools. In eukaryotic cells, CRISPR-Cas13 initially seemed to exclusively cleave the target RNA and consequently, CRISPR-Cas13 has been adopted as a specific RNA knockdown tool. Recently, several groups have reported unexpected toxicity or collateral cleavage when using CRISPR-Cas13 in eukaryotic cells, which seems difficult to reconcile with the reported target specificity. To understand these seemingly contradicting findings, we explored the collateral cleavage activity of six Cas13 systems, and show that only the most active ortholog in vitro, LbuCas13a, exhibits strong collateral RNA cleavage activity in human cells. LbuCas13a displayed collateral cleavage in all tested cell lines, targeting both exogenous and endogenous transcripts and using different RNP delivery methods. Using Nanopore sequencing, we found that cytoplasmic RNAs are cleaved without bias by LbuCas13a. Furthermore, the cleavage sites are highly specific and often present in Uracil containing single stranded RNA loops of stem-loop structures. In response to collateral RNA cleavage, cells upregulate stress and innate immune response genes and depending on target transcript levels, RNA degradation resulted in apoptotic cell death. We demonstrate that LbuCas13a can serve as a cell selection tool, killing cells in a target RNA specific manner. As such, CRISPR-Cas13 is a promising new technology that might be useful in anti-tumor applications.

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A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13

Cited by 14 publications

References 76 publications

Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity

Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

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