Note: an updated version of this repository is available at https://github.com/QilabGitHub/CRISPR-PACMAN-pool-generation. The new repository includes working examples.

Our updated code pipeline has been run on a large number of viral families and groups. You can access these pre-generated guide RNA pools at http://crispr-pacman.stanford.edu/

nCov2019_Guide_Design

1. Design effective and specific crRNAs for nCov2019, MERS and SARS

Detect 22nt sequences in conserved region (Perfect match COVID-19 <=1 mismatch SARS/MERS)

python horizontal-pool/main.py aligned_sequences.fasta -o ./output_data

Remove Off-target in human transcriptome by using bowtie

bowtie -a -S Human_GRCh38_Transcriptome_BowtieIndex nCov_22nt_seqs.fq nCov_22nt_seqs_OffTarget_HG38_RNA.sam -v 2
python Summarize_OffTarget.py nCov_22nt_seqs_OffTarget_HG38_RNA.sam Total_RNA_Number 0 nCov_22nt_seqs_OffTarget_Summary.txt

Generate crRNA space sequences and filter "TTTT"

python generate_crRNA_spacer.py nCov_22nt_seqs_NoOffTarget.txt colnum_22nt_Seqs Colnum_22nt_Seqs_Names Final_crRNA.output

2. Design pan-coronavirus crRNAs

Collect 22nt unique sequences for all known CoV genomes and generate a minimum pool of 22 crRNAs for targeting all CoV genomes

python minipool/main.py sequences.fasta -o ./output_data

Remove Off-target in human transcriptome by using bowtie

bowtie -a -S Human_GRCh38_Transcriptome_BowtieIndex nCov_22nt_seqs.fq nCov_22nt_seqs_OffTarget_HG38_RNA.sam -v 2
python Summarize_OffTarget.py nCov_22nt_seqs_OffTarget_HG38_RNA.sam Total_RNA_Number 0 nCov_22nt_seqs_OffTarget_Summary.txt

Generate crRNA space sequences and filter "TTTT"

python generate_crRNA_spacer.py nCov_22nt_seqs_NoOffTarget.txt colnum_22nt_Seqs Colnum_22nt_Seqs_Names Final_crRNA.output