Collateral activity of the CRISPR/RfxCas13d system in human cells

Abstract

CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.

Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins

“…Knowing this, we evaluated the targeting capabilities of the Hi-Fi RfxCas13d variants, RfxCas13d-N1V7, -N2V7 and -N2V8, as well as DiCas7-11, in HEK293T cells transiently transfected with the mATXN2-T2A-EGFP-encoding reporter plasmid alongside an mCherry-encoding reporter, whose fluorescence was used as a proxy for collateral targeting. Consistent with prior studies demonstrating that targeting highly expressed reporter transcripts in mammalian cells can lead to robust collateral effects [50][51][52][53][54] , we found that, when programmed to target mATXN2-T2A-EGFP, the native RfxCas13d protein reduced mCherry fluorescence by ~40-60% (P < 0.001; Fig. 6a and Supplementary Fig.…”

“…We next evaluated if RfxCas13d could knock down the endogenous mATXN2 transcript and if its targeting induced collateral effects in cells, as RfxCas13d-mediated trans-cleavage can lead to the degradation of non-specific RNA [50][51][52][53][54] . To answer this first question, we transfected Neuro2A mouse neuroblastoma cells with RfxCas13d and crRNA-10, the most efficient mATXN2-targeting crRNA identified from our initial screen.…”

“…To measure potential collateral effects, we transfected Neuro2A cells with RfxCas13d and crRNA-10 alongside an mCherry-encoding surrogate reporter, whose fluorescence was used as proxy for collateral activity (e.g., by this method [50][51][52][53][54] collateral trans-cleavage by RfxCas13d is expected to decrease mCherry fluorescence). Relative to cells transfected with mCherry alongside an inert carrier plasmid, we measured no significant decrease in the percentage of mCherry + cells or mCherry fluorescence intensity in cells transfected with RfxCas13d and crRNA-10 (P > 0.05 for both, Supplementary Fig.…”

“…Upon binding to its target RNA, RfxCas13d -like other Cas13 effectors -can collaterally cleave non-target RNAs 50,[52][53][54] . Because of this, high-fidelity (Hi-Fi) forms of the protein have been developed that possess a reduced capacity to trans-cleave non-target RNAs, while still retaining the ability to cleave target RNA 72 .…”

“…The extent with which Cas13 enzymes can collaterally cleave RNAs can correlate with the expression threshold of the target transcript, with targeting of an overexpressed mRNA thought to result in more robust collateral effects [50][51][52][53] . Knowing this, we evaluated the targeting capabilities of the Hi-Fi RfxCas13d variants, RfxCas13d-N1V7, -N2V7 and -N2V8, as well as DiCas7-11, in HEK293T cells transiently transfected with the mATXN2-T2A-EGFP-encoding reporter plasmid alongside an mCherry-encoding reporter, whose fluorescence was used as a proxy for collateral targeting.…”