DOI: 10.5061/dryad.sqv9s4n9w
title: tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

description: 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.
description: 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
description: # 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,
 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 | File name | Sample description | Experimental group |
 | :------------------------------------ |
 :---------------------------------------------------------------------------------------------------------------------------------------- | :----------------- | | NT1\_5min\_S4\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, non-targeting cells, replica 1 | non-targeting | | NT2\_5min\_S5\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, non-targeting cells, replica 2 | non-targeting | | NT3\_5min\_S6\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, non-targeting cells, replica 3 | non-targeting | | T1\_5min\_S1\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, targeting cells, replica 1 | targeting | | T2\_5min\_S2\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, targeting cells, replica 2 | targeting | | T3\_5min\_S3\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 5 min post induction, targeting cells, replica 3 | targeting | | NT1\_60min\_S16\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 1, forward reads | non-targeting | | NT1\_60min\_S16\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 1, reverse reads | non-targeting | | NT2\_60min\_S17\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 2, forward reads | non-targeting | | NT2\_60min\_S17\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 2, reverse reads | non-targeting | | NT3\_60min\_S18\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 3, forward reads | non-targeting | | NT3\_60min\_S18\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 3, reverse reads | non-targeting | | T1\_60min\_S13\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 1, forward reads | targeting | | T1\_60min\_S13\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 1, reverse reads | targeting | | T2\_60min\_S14\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 2, forward reads | targeting | | T2\_60min\_S14\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 2, reverse reads | targeting | | T3\_60min\_S15\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 3, forward reads | targeting | | T3\_60min\_S15\_R2\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 3, reverse reads | targeting | | d10\_NT1\_60min\_S4\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 1, forward reads | non-targeting | | d10\_NT1\_60min\_S4\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 1, reverse reads | non-targeting | | d10\_NT2\_60min\_S5\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 2, forward reads | non-targeting | | d10\_NT2\_60min\_S5\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 2, reverse reads | non-targeting | | d10\_NT3\_60min\_S6\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 3, forward reads | non-targeting | | d10\_NT3\_60min\_S6\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, non-targeting cells, replica 3, reverse reads | non-targeting | | d10\_T1\_60min\_S1\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 1, forward reads | targeting | | d10\_T1\_60min\_S1\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 1, reverse reads | targeting | | d10\_T2\_60min\_S2\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 2, forward reads | targeting | | d10\_T2\_60min\_S2\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 2, reverse reads | targeting | | d10\_T3\_60min\_S3\_R1\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 3, forward reads | targeting | | d10\_T3\_60min\_S3\_R2\_001.fastq.gz | *E. coli* Δ10 cells encoding *L. shahii* Type VI CRISPR-Cas locus, 60 min post induction, targeting cells, replica 3, reverse reads | targeting | | M13\_NT1\_S10\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, non-targeting cells, replica 1 | non-targeting | | M13\_NT2\_S11\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, non-targeting cells, replica 2 | non-targeting | | M13\_NT3\_S12\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, non-targeting cells, replica 3 | non-targeting | | M13\_T1\_S7\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, targeting cells, replica 1 | targeting | | M13\_T2\_S8\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, targeting cells, replica 2 | targeting | | M13\_T3\_S9\_R1\_001.fastq.gz | *E. coli* C3000 cells encoding *L. shahii* Type VI CRISPR-Cas locus infected with M13 phage, targeting cells, replica 3 | targeting | | Ish\_10\_S23\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 1, forward reads | non-targeting | | Ish\_10\_S23\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 1, reverse reads | non-targeting | | Ish\_11\_S24\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 2, forward reads | non-targeting | | Ish\_11\_S24\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 2, reverse reads | non-targeting | | Ish\_12\_S25\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 3, forward reads | non-targeting | | Ish\_12\_S25\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in absence of target transcript, replica 3, reverse reads | non-targeting | | Ish\_7\_S20\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 1, forward reads | targeting | | Ish\_7\_S20\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 1, reverse reads | targeting | | Ish\_8\_S21\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 2, forward reads | targeting | | Ish\_8\_S21\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 2, reverse reads | targeting | | Ish\_9\_S22\_R1\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 3, forward reads | targeting | | Ish\_9\_S22\_R2\_001.fastq.gz | *E. coli* total RNA incubated with LshCas13a:CRISPR-RNA ribonucleoprotein in presence of target transcript, replica 3, reverse reads | targeting | 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