The double life of CRISPR–Cas13

Bot, Jorik F; Oost, John van der; Geijsen, Niels

doi:10.1016/j.copbio.2022.102789

Cited by 26 publications

(23 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We noted that commonly used collateral cleavage detection methods, namely RT-qPCR and RNA-seq, are not suitable for the detection and quantification of collateral cleavage, because they are relative quantification methods. RT-qPCR relies on the stable expression of an intrinsic reference RNA [55]. In the case of Cas13-mediated collateral cleavage such reference RNA is itself subject to degradation.…”

Section: Discussionmentioning

confidence: 99%

“…Since previous reports demonstrated that Cas13 does not display collateral cleavage in certain cell lines [55], we explored whether collateral RNA cleavage was perhaps cell-type dependent. In addition, we examined if LbuCas13a collateral cleavage could also be induced targeting endogenous transcripts and using a different RNP delivery method.…”

Section: Exploring the Requirements For Cas13 Collateral Cleavage In ...mentioning

confidence: 99%

“…We delivered the five remaining Cas13 proteins either with a non-targeting or a dEGFP targeting guide RNA in HAP1 cells stably overexpressing a mutant, fluorescenceinactivated EGFP [54] (dEGFP Y66S , referred to as dEGFP from hereon), and isolated RNA fifty minutes after RNP transduction. While RT-qPCR has been commonly used to assess for collateral RNA cleavage [55], this approach may not be suitable, as it depends on the assumption that the expression of a reference transcript is unaffected by the treatment. If Cas13 indeed behaves as a non-specific RNase, and also degrades the reference transcripts, this assumption no longer holds.…”

Section: Lbucas13a Exhibits Collateral Cleavage In Human Cellsmentioning

confidence: 99%

See 2 more Smart Citations

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

Bot

Zhao

Kammeron

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Exploring the Requirements For Cas13 Collateral Cleavage In ...mentioning

confidence: 99%

Section: Lbucas13a Exhibits Collateral Cleavage In Human Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

Bot

Zhao

Kammeron

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Second, RNAi usually shows a 70-90% reduction in circRNA expression but Cas13 usually shows much decreased levels of circRNA suppression (40-60%) making it difficult to identify phenotypes. Third, collateral damage activity 47 induces a large number of off target effects and it is difficult to determine if an observed phenotype is a result of the on target or off target Cas13 effect. Although, the recent development of Cas7-11 with no observed collateral RNA cleavage may be used in future studies 48 , the current low cutting efficiency of the Cas7-11 system will need to be improved to enable functional readouts.…”

Section: Discussionmentioning

confidence: 99%

Systematic loss of function screens identify pathway specific functional circular RNAs

Liu

Neve

Perlaza-Jiménez

et al. 2022

Preprint

View full text Add to dashboard Cite

Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are produced by RNA back-splicing. A small number of circRNAs have been implicated as functional, however, we still lack systematic understanding of cellular processes and signalling pathways that are regulated by circRNAs. A major gap, in understanding circRNAs functions is the ability to define pathways that are regulated by circRNAs. Here, we generated a pooled shRNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at low to high levels in humans. We used this library for loss of function proliferation screens in a panel of 18 cancer cell lines from four tissue types that harbour mutations leading to constitutive activity of defined pathways. Using this dataset, we identify context specific and non-specific circRNAs. We validated these observations with a secondary screen and uncovered a role forcircRERE, a cell essential circRNA that regulates ferroptosis. Furthermore, we characterised the functional roles of pathway-specific circRNA,circSMAD2, a novel regulator of the WNT pathway andcircMTO1, a regulator of MAPK signalling in aPTENdependent manner. Our work sheds light on molecular pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function in human cells.

show abstract

“…Cas13 is one such system that originally evolved as an RNA antiviral in prokaryotes 11 , and has surfaced as programmable RNA-targeting ribonucleases 11 . The high efficiency of programmable Cas13 has been demonstrated in human cells 12 , zebrafish 13 , mice 14 , flies 15,16 and plants 17 , but the high on-target activity is natively coupled with collateral cleavage of bystander RNAs 18 . Although this collateral cleavage may limit the potential of Cas13 for therapeutic applications, we hypothesized that it could be leveraged in mosquitoes to combat viral infections, providing a basis for the development of flexible antiviral technologies.…”

Section: Introductionmentioning

confidence: 99%

Engineered Antiviral Sensor Targets Infected Mosquitoes

Benetta

López-Denman

et al. 2023

Preprint

View full text Add to dashboard Cite

Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

show abstract

The double life of CRISPR–Cas13

Cited by 26 publications

References 68 publications

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

Collateral RNA cleavage by CRISPR-Cas13 allows selective cell elimination

Systematic loss of function screens identify pathway specific functional circular RNAs

Engineered Antiviral Sensor Targets Infected Mosquitoes

Contact Info

Product

Resources

About