Article-Genomic Labeling

Spuriously transcribed RNAs from CRISPR-sgRNA expression plasmids scaffold biomolecular condensate formation and hamper accurate genomic imaging

Our results indicated that false-positive foci do not result from nonspecific accumulation of sgRNAs as previously speculated. Instead, they pointed to a previously undescribed role of cryptic plasmid transcripts, which arise from spurious transcriptional activities occurring within plasmid DNA regions other than the intended promoter-gene cassette (i.e. transcription unit). These unintended transcripts are sequestered by paraspeckle proteins FUS, SFPQ and PSPC1 rather than accumulating around their producer plasmids, leading to false-positive foci formation. Based on these findings, we presented an easy-to-adapt strategy to minimize the generation of false-positive signals.

Abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging tools that utilize fluorescently tagged single-guide RNAs (sgRNAs) have enabled versatile analysis of the dynamics of single genomic loci, but the accuracy may be hindered by nonspecific subnuclear probe accumulation, generating false-positive foci in cell nuclei. By examining the subcellular localizations of sgRNA expression plasmids, their RNA transcripts, and several RNA-binding proteins, we found that spuriously transcribed (cryptic) transcripts, produced by sgRNA expression plasmids, are the major contributors of false-positive signals, independent of sgRNA scaffold design or effector probe (i.e. RNA aptamer- or oligonucleotide-based probes) used. These transcripts interact with the paraspeckle core proteins, but not with the sgRNA expression plasmids or the paraspeckle RNA scaffold NEAT1_2, to form nuclear bodies that display liquid-like properties including sphericality, fusion competence, and sensitivity to 1,6-hexanediol. Transfecting sgRNA transcription units (i.e. sgRNA expression cassettes), lacking the plasmid backbones, reduces false-positive signals and enhances genomic imaging accuracy. Overall, this study unveils previously undescribed activities of cryptic plasmid transcripts and presents an easy-to-adapt strategy that can potentially improve the precision of CRISPR-based imaging systems that implement fluorescently tagged sgRNAs.

See full-text: https://doi.org/10.1093/nar/gkaf192

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CRISPR imaging Cryptic transcripts Liquid like structure

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