We present a combinatorial indexing method, PerturbSci-Kinetics, for capturing whole transcriptomes, nascent transcriptomes and single guide RNA (sgRNA) identities across hundreds of genetic perturbations at the single-cell level. Profiling a pooled CRISPR screen targeting various biological processes, we show the gene expression regulation during RNA synthesis, processing and degradation, miRNA biogenesis and mitochondrial mRNA processing, systematically decoding the genome-wide regulatory network that underlies RNA temporal dynamics at scale.
© 2023. The Author(s).

Here we described PerturbSci-Kinetics , a novel combinatorial indexing method for capturing three-layer single-cell readout ( i . e ., whole transcriptome, nascent transcriptome, sgRNA identities) across hundreds of genetic perturbations. Through PerturbSci-Kinetics profiling of pooled CRISPR screens targeting a variety of biological processes, we were able to decipher the complexity of RNA regulations at multiple levels ( e . g ., synthesis, processing, degradation), and revealed key regulators involved in miRNA and mitochondrial RNA processing pathways. Our technique opens up the possibility of systematically decoding the genome-wide regulatory network underlying RNA temporal dynamics at scale and cost-effectively.