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Engineered, nucleocytoplasmic shuttling Cas13d enables highly efficient cytosolic RNA targeting
Monday, 2024/04/22 | 08:17:07

Christoph GruberLea KrautnerValter BergantVincent GrassZhe MaLara RheinemannAriane KrusFriederike ReinhardtLyupka MazneykovaMarianne Rocha-HaslerDong-Jiunn Jeffery TruongGil Gregor WestmeyerAndreas PichlmairGregor EbertFlorian Giesert & Wolfgang Wurst

Cell Discovery 10, Article number: 42 (2024) 

Published: 12 April 2024


CRISPR/Cas13 systems are programmable tools for manipulating RNAs and are used in a variety of RNA-targeting applications. Within the Cas13 family, Cas13d is the most active subtype in mammalian cells. Recently, Cas13d was harnessed as an antiviral against diverse human RNA viruses. However, Cas13d is barely active in the cytosol of mammalian cells, restricting its activity to the nucleus, which limits applications such as programmable antivirals. Most RNA viruses replicate exclusively in the cytosol, suggesting that current Cas13d-based antivirals rely on uncontrolled nuclear leakage and are therefore limited in their efficiency7.


Here, we show that the nuclear localization of Cas13d crRNAs is the fundamental cause of Cas13d’s nuclear preference. To address this limitation, we engineered nucleocytoplasmic shuttling Cas13d (Cas13d-NCS). Cas13d-NCS transfers nuclear crRNAs to the cytosol, where the protein/crRNA complex binds and degrades complementary target RNAs. We screened various designs of shuttling proteins and characterized multiple design parameters of the best-performing system. We show that Cas13d-NCS is superior for degrading mRNAs and a self-replicating RNA derived from the Venezuelan equine encephalitis (VEE) RNA virus. Ultimately, we harnessed Cas13d-NCS to completely block the replication of various SARS-CoV-2 strains. Cas13d-NCS, therefore, enables the rational manipulation of the subcellular localization of a CRISPR system.


In our study, we demonstrated that conventional Cas13d applications are limited to the nucleus due to crRNA localization. In contrast, Cas13d-NCS transports crRNAs out of the nucleus by shuttling between the nucleus and cytosol. Previous attempts to redirect the Cas13d system to the cytosol by fusing an NES did not take crRNA localization into account or relied on uncontrolled nuclear leakage of the crRNA/protein complex4,5,7. Other CRISPR systems, dependent on nuclear-transcribed gRNAs, could benefit from the described crRNA transport framework as well by applying similar engineering principles to these systems. In summary, we envision that Cas13d-NCS will unleash the full potential of RNA targeting with subcellular precision and enable the development of novel molecular tools and therapies for RNA-related diseases.


See https://www.nature.com/articles/s41421-024-00672-1


Fig. 1: Engineering of Cas13d-NCS for efficient cytosolic RNA and antiviral targeting.

a Schematic illustration of the expression and localization of Cas13d protein and crRNA in mammalians. Cas13d-NES has one C-terminal NES motif, corresponding to v5. b RNA FISH staining of the crRNA along with staining for GAPDH mRNA in cells expressing nuclear Cas13d-NLS (3xNLS, corresponds to v1 in f). Scale bar: 15 μm. c Schematic illustration of nucleocytoplasmic shuttling Cas13d to transport crRNAs to the cytosol. d RNA FISH staining of pol III expressed crRNAs, co-expressed with either nuclear (v1) or shuttling Cas13d (v3). Scale bar: 10 μm. e Quantification of cytosolic/total crRNA staining intensity for co-expressed nuclear and shuttling Cas13d. Unpaired Student’s t-test, ****P < 0.0001, mean ± s.d. for n = 50 cells. f Illustration of Cas13d shuttling constructs with varying NLS/NES motifs. g Quantification of cytosolic/total Cas13d protein staining intensity for different variants v1–v5 (n = 50 cells). h Comparison of nanoluciferase knockdown for Cas13d variants v1–v5. Unpaired Student’s t-test, ****P < 0.0001, mean ± s.d. for n = 6 biological replicates. i Schematic illustration of VEE reporter replicon. j Fluorescence imaging of replicon-expressing cells after short- and long-term cultivation. Scale bar: 30 μm. k Comparison of replicon targeting efficiency between nuclear and shuttling Cas13d-NCS using flow cytometry. Unpaired Student’s t-test, **P < 0.01; ****P < 0.0001, mean ± s.d. for 9 crRNAs, each measured in n = 3 biological replicates. l Schematic illustration of SARS-CoV-2-GFP reporter virus and viral transcriptome, targeted by a single 3’UTR crRNA. m Live measurement of SARS-CoV-2-GFP viral load by integrated GFP intensity under different targeting conditions (mean ± s.d., n = 4 biological replicates). n RNA-seq analysis of SARS-CoV-2 (Delta) subgenomic mRNA expression after 48 h of treatment with Cas13d-NCS and a non-target or 3’UTR target crRNA (n = 3 biological replicates).


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