A Remote Control for RNA-Targeting CRISPR

Review written by: Megan Mauriello

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic antiviral immunity system enabled by various Cas effector proteins. Cas13 specifically targets RNA, cleaving ssRNAs complementary to a guide RNA. Cas13 has applications in programmable RNA cleavage, detection, and imaging. However, existing Cas13 systems have weaknesses due to being constitutively active or by virtue of being dual-component systems. Additionally, with Cas13 systems, it is important to control Cas13 and target RNA cleavage in order to mitigate trans-cleavage, which can be lethal to cells.

Ruminococcus flavefaciens Cas13d (RfxCas13d) works by binding to a host CRISPR RNA (crRNA), forming a binary complex, which then binds to the target RNA, forming a ternary complex, and resulting in target RNA cleavage.

Figure. a, b Comparison of the mechanism of a wild-type RfxCas13d cleavage (in (a)) and an allosterically controlled RfxCas13d via light-sensitive domain insertion (in (b)). c Schematic of the workflow for generating RfxCas13d-AsLOV2 insertion library and incorporating them into landing pad reporter cell lines for selections. d Coverage of random AsLOV2 insertion sites in between amino acid residues within RfxCas13d through next-generation sequencing analysis. e Composition of a landing pad reporter cell line. This cell line contains a single recombination site with the BFP gene in frame for negative selection. This recombination site acts as a landing pad for the incorporation of the RfxCas13d-AsLOV2 insertion library. The landing pad cell line also contains a reporter EGFP gene and a crRNA targeting EGFP mRNA. f Successful incorporation of the RfxCas13d-AsLOV2 insertion library results in displacement of the BFP gene by mCherry. BFP- negative and mCherry-positive cell population was sorted via fluorescence-activated cell sorting (FACS). EGFP knockdown was confirmed in this sorted subpopulation.

RfxCas13d was used as a basis for a random AsLOV2-domain insertion library with DIP-Seq. AsLOV2 is a photoswitchable domain. Screening of insertion variants and incorporation into a mammalian cell line led to the development of a RfxCas13d-AsLOV2 library which could then be screened for EGFP knockdown in light and dark conditions. This screen revealed selection against constitutive Cas13d activity, likely due to lethal and/or sublethal trans-cleavage. Next Generation Sequencing of variants before and after cell incorporation revealed a variant with an insertion at QK634, which was more active in the dark than in the blue light. AlphaFold 3 modeling suggested that QK634 was an allosteric activation site. This QK634-insertion variant was dubbed OptoCas13d-off. Light inactivation of activity was most robust at a specific range of Cas13d expression level. To identify the process(es) which were inactivated by blue light, OptoCas13d-off was introduced into Escherichia coli. A combination of electrophoretic mobility shift assays and utilization of catalytically inactive Cas13d determined that light likely inactivated RfxCas13d-cRNA-target ternary complex formation and target RNA cleavage, but not RfxCas13d-cRNA binary complex formation.

Replacing the AsLOV2-domain insertion in OptoCas13d-off with either a LightR or UniRapR domain substituted light inactivation of knockdown activity with light activation or rapamycin activation of activity, respectively. The LightR insertion construct, OptoCas13d-on, was validated to only be active under blue light, to also work on endogenous transcripts, and was even reversible when dark conditions were restored. Similarly, the UniRapR-insertion construct, ChemoCas13d, was validated to work on endogenous transcripts, with genomic integration site and expression activity playing large roles.

Overall, this study establishes a pipeline for isolating cell lines with tightly controlled activity of a Cas13d construct. Although OptoCas13d-off, OptoCas13d-on, and ChemoCas13d could benefit from some optimization, this approach and these resulting constructs represent a significant step in the engineering of CRISPR-Cas13 systems for various applications. Constructs from this pipeline avoid most of the disadvantages of constitutively active or dual-component systems.

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Zhu L, Nguyen LT, Bell AG, Krebel T, Gillmann KM, Cao Q, Oatman H, Hariri J, Möglich A, Myhrvold C, Toettcher JE. Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot. Nat Commun. 2026 Jun 3;17(1):7146. doi: 10.1038/s41467-026-73645-5. PMID: 42236706; PMCID: PMC13396516.