https://doi.org/10.5061/dryad.sqv9s4n9w

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, purified LshCas13a proteins loaded with either targeting or non-targeting CRISPR-RNA were incubated with the target transcript in the presence of total RNA isolated from E. coli cells.

Subsequently, the RNA molecules underwent sequencing using high-throughput techniques. Each experiment was performed in three biological replicates. In the experiments with E. coli Δ10 strain, E. coli C3000 strain at 60 min post induction and in in vitro experiments with total RNA sequencing was performed in paired ends mode. 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.

Description of the data and file structure

The scripts used for the analysis could be found in the archive LshCas13a_RNA_cleavage-master.zip. For the analysis, the raw fastq files should be put in the Data/Raw catalogs in the corresponding directory.

Each directory contains data and scripts for the particular experiment:

• LshCas13a_C3000 - RNA-Seq of total RNA extracted from E. coli C3000 cells carrying activated/nonactivated LshCas13a enzyme;
• LshCas13a_d10LVM - RNA-Seq of total RNA extracted from E. coli d10 cells carrying activated/nonactivated LshCas13a enzyme;
• LshCas13a_in_vitro_total_RNA - RNA-Seq of total RNA extracted from E. coli C3000 cells after in vivo incubation with activated/nonactivated LshCas13a enzyme;
• LshCas13a_M13_infection - RNA-Seq of total RNA extracted from E. coli C3000 cells harboring Type VI spacer against M13 phage and infected with M13 phage;

Each directory contains the following subdirectories:

• Data - directory containing the raw reads data;
• Annotations - directory containing GFF tables with genomic features;
• Alignments - directory containing alignments produced with read_mapping.sh script;
• Reference_sequences - directory containing FASTA files of sequences used for reads mapping;
• Scripts - directory containing scripts for data processing;
• Results - directory containing the results of data processing.

The "Results" directory contains the following subdirectories:

• Tables
  • Ends_counts - contains files with coordinates of 5' ends of fragments;
  • Fragment_coords - contains files with coordinates of fragments (SeqID - Fragment_start - Fragment_end - Strand)
  • Merged_ends_counts - contains tables with 5' ends counts derived from samples designeted for comparison
  • Read_pairs_TABs - contains tables with coordinates of read pairs.
• WIG_files - contains wig-files with 5' ends coverage.

The "Scripts" directory contains a set of scripts for the data processing. There is a "basic" set of scripts which is common for all experiments:

• raw_data_processing.sh - performs reads quality assessment, removes adapters and discards low-quality reads.
  • Requirements:
    • fastqc, trimmomatic
• read_mapping.sh - maps paired-end reads to the reference sequences. Since the SAM alignments file are quite large, the output data is compressed using gzip.
  • Requirements:
    • bowtie2
• return_fragment_coords_table.py - receives alignment files (in gzipped SAM format) and generates all tables deposed in "Result-Tables" directory (except "Merged_ends_counts") and produces WIG files with 5' ends coverage;
  • Requirements:
    • python3 with gzip and pandas modules
• merge_ends_count_tables.py - combines 5' ends counts tables from different tables into one table
  • Requirements:
    • python3 with pandas, gzip, re and functools modules
• TCS_calling.R - performs statistical test producing table with the position, logFC and p-value values.
  • Requirements:
    • R with dplyr, data.table, tidyr and edgeR modules