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 compared between targeting and non-targeting samples.

Methods
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 during library preparation. Samples were purified using the Zymo Research Oligo Clean and Concentrator kit. Library preparation for RNA sequencing was done using the NEBNext Multiplex Small RNA Library Prep Set for Illumina according to manufacturer’s protocol. BluePippin size selection was done using 2% agarose gel cassette (Sage Science) to select for 100 - 600 bp products. QC at each step was carried out by both Qubit and fragment analyzer. RNA sequencing was performed using Illumina NextSeq High-Output kit 2 × 35 bp paired end read length.

Raw RNA sequencing reads were filtered by quality with the simultaneous adapters removal using trimmomatic v. 0.36. The exact parameters of trimmomatic run could be found in raw_data_processing.sh file.

Processed reads were mapped onto reference sequences (RefSeq: NC_000913.3 supplemented with pC002 and pC008 plasmids in case of nontargeting samples and NC_000913.3 supplemented with pC003_RFP_spacer and pC008 plasmids in case of targeting samples) using bowtie2 v. 2.3.4.3 producing corresponding SAM files. In case of the analysis of data obtained from in vitro experiments on isolated E. coli total RNA reads were mapped only onto the NC_000913.3 sequence. The exact parameters of bowtie2 run could be found in read_mapping.sh file.For each nucleotide position of each strand of reference sequences the number of 5’ ends of aligned fragments were counted producing corresponding tables (see return_fragment_coords_table.py file for details).

The obtained tables were joined using merge_ends_count_tables.py script. The differences between the numbers of mapped 5’ ends in targeting and nontargeting samples were analyzed using edgeR package v. 3.26.3. The features (here, strand specific nucleotide positions) with low counts were excluded from the analysis. The TMM normalization method implemented in edgeR was applied. Next, the edgeR likelihood ratio test was performed. The obtained p-values were corrected using Benjamini-Hochberg method, and the result tables containing analyzed features with assigned log2FC and adjusted p-values were written to separate files (see TCS_calling.R file for details). The obtained trascript cleavage sites were overlapped with annotated genomic features using GenomicRanges package. Genome_build: NC_000913.3