This project is about mining Cas13 proteins from metagenomic data and RNA off-target detection of Cas13/Cas13-ADAR2dd.

a. Identify the Cas13 proteins from metagenomic samples.

a.1. Install the “prodigal.linux” , "bedtools" and “pilercr” in the default environment. bedtools: https://sourceforge.net/projects/bedtools/ pilecr: http://www.drive5.com/pilercr/

a.2. run “perl 0.Cas-Finder $sample.fasta” to obtain the Cas proteins and generate "$sample.pep.cas.fasta" file.

a.3. run " perl 1.Cas13-Finder.pl $sample.pep.cas.fasta" to otain the Cas13 proteins and generate "$sample.pep.cas.RxxxxH.fa" file.

a.4. Multiple alignment of Cas13 proteins with mafft.

mafft --maxiterate 1000 --thread 12 --globalpair Cas13.fa > Cas13.mafft.fasta

b. RNAseq off-Target analysis of Cas13.

b.1. align the RNAseq to reference genome

hisat2 -p 18 --dta-cufflinks -q -x GRCh38.genome.fa -1 {$sample}_1.clean.fq.gz -2 {$sample}_2.clean.fq.gz -S $sample.sam

samtools view -Su -q 30 $sample.sam | samtools sort -@ 18 - > $sample.sorted.bam

samtools index $sample.sorted.bam

b.2. Calculate the read counts.

htseq-count -f bam $sample.sorted.bam GRCh38.gtf > $sample.HTSeq.out

b.3. Convert the read counts into FPKM values.

perl HTSeq2FPKM.pl $sample.HTSeq.out GRCh38.gtf

b.4. Calculate differently expressed gene using DEseq2

perl ~/path/trinityrnaseq-Trinity-v2.8.5/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix gene_count_matrix.csv.xls --method DESeq2 --output DESeq2_out --

samples_file sample.txt --contrasts contrast.txt --dispersion 0.04

perl 4.get_DEG-DESeq2.pl gene_count_matrix.csv.xls.Control_vs_Treat.DESeq2.DE_results

b.5. Predict the off-target site of Hg38 genome and transcriptome of the spacers with no more than eight mismatches.

perl Mismatch-search.pl Spacer.fa Hg38.genome.fa 8

perl OffTarget_gene.pl Spacer.Mismatch.fa Hg38.gtf

c. RNAseq off-target analysis of RNA base editor.

c.1. align the RNAseq to reference genome

hisat2 -p 18 --dta-cufflinks -q -x GRCh38.genome.fa -1 $sample_1.clean.fq.gz -2 $sample_2.clean.fq.gz -S $sample.sam

samtools view -Su $sample.sam | samtools sort -@ 18 - > $sample.sorted.bam

c.2. Install the REDItool fom "https://sourceforge.net/projects/reditools/" .

python ~/software/anaconda3/envs/py2/bin/REDItoolDenovo.py -o $sample.REDtools -i $sample.sorted.bam -f GRCh38.genome.fa -t 24 -e -d -l -U [AG,TC,CT,GA] -p -u -m60 -T5-5 -W -v 1 -n 0.0