tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

In this study, we conducted an analysis of RNA cleavage products mediated by the Type VI CRISPR-Cas system from the bacterium Leptotrichia shahii. Previous research has demonstrated that the LshCas13a effector protein, when loaded with CRISPR-RNA, exhibits collateral RNase activity upon recognizing the target transcript. To identify the products resulting from RNA cleavage mediated by the activated Type VI CRISPR-Cas system, we isolated total RNA samples from E. coli cells expressing either activated (targeting samples) or non-activated (non-targeting samples) LshCas13a effectors. Subsequently, the RNA molecules underwent sequencing using high-throughput techniques. Each experiment was performed in three biological replicates. The acquired data underwent processing to eliminate technical sequences and low-quality reads, followed by alignment to the reference genomic sequences. Subsequently, the counts of 5' end positions of the sequenced fragments were determined, and these counts were ..., Total RNA was isolated from cells pelleted at 1 hour post RFP induction. Cell lysis was done using Max Bacterial Enhancement Reagent (Invitrogen) for 4 min and then with TRIzol reagent (Invitrogen) for 5 min. RNA was extracted by chloroform and precipitated with isopropanol. RNA pellets were washed with 70% ethanol and then dissolved in nuclease free water and then treated with Turbo DNA-free kit (Invitrogen).
Total RNA samples were treated with MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen) for rRNA depletion prior to library preparation. To obtain both primary (5’ PPP) and processed (5’ P/5’ OH) transcripts, RNA samples were treated with RNA 5' Pyrophosphohydrolase (RppH) (NEB) for 30 min at 37C which removes pyrophosphate from 5’ end from triphosphorylated RNA to leave a 5’ monophoshphate RNA. Fragmentation was carried out by sonication using the Covaris protocol to obtain fragments of 200 nt size. T4 PNK (NEB) treatment was done to obtain 5’P ends for adapter ligation duri..., , # tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

## **Abstract**

In this study, we conducted an analysis of RNA cleavage products mediated by the Type VI CRISPR-Cas system from the bacterium *Leptotrichia shahii*. Previous research has demonstrated that the LshCas13a effector protein, when loaded with CRISPR-RNA, exhibits collateral RNase activity upon recognizing the target transcript. To identify the products resulting from RNA cleavage mediated by the activated Type VI CRISPR-Cas system, we isolated total RNA samples from *E. coli cells* expressing either activated (targeting samples) or non-activated (non-targeting samples) LshCas13a effectors. In the experiments with the targeting of plasmid-borne transcript, expression of the target was induced by the addition of the anhydrotetracycline. In the experiments with phage infection, cells encoding or not encoding Type VI spacer targeting M13 transcript were infected with M13 phage. In *in vitro* experiments, pur...