EXCAVATE-HT Examples

Example run: Generating a paired dual-gRNA library

Generating paired dual-gRNAs targeting heterozygous SNVs in an individual cell-line in the MFN2 locus, for the SpCas9 enzyme, paired via “random” method (all gRNA targeting different SNPs paired together) in the locus. Off-targets enabled, –download-hg38-index will automatically download required hg38 indexes for off-targets analysis.

excavate-ht generate \
  --vcf cellline.vcf.gz \
  --var_type cell-line \
  --chrom_fa chrom1.fasta \
  --locus chr1:11975541-12019490 \
  --cas SpCas9 \
  --pairing-method r \
  --off-targets \
  --download-hg38-index \
  --summary \
  -o paired_gRNA_output_folder

Example run: Generating a single-gRNA library, editing it, then pairing to create a dual-gRNA library

Generaing single-gRNAs targeting heteroyzgous SNVs in an individual cell-line and common SNPs present in the 1000 genomes project, in the MFN2 locus, for a custom 5prime PAM sequence. Off-targets enabled, pre-built genome and chromosome index file prefixes provided using –genome-index-prefix and –chrom-index-prefix flags

excavate-ht generate \
  --vcf cellline.vcf.gz,1000genomes_chrom1.vcf.gz \
  --var_type cell-line,population \
  --chrom_fa chrom1.fasta \
  --locus chr1:11975541-12019490 \
  --pam-list TRTV \
  --orient 5prime \
  --off-targets \
  --genome-index-prefix /path/to/hg38genome_index_prefix/genome_bt1 \
  --chrom-index-prefix /path/to/chrom1_index_prefix/genome_bt1 \
  --summary \
  -o single_gRNA_output_folder

This sgRNA library can now be manually edited. gRNA with many off-target sites, as revealed by the –off-targets analysis option, can be filtered out, and screening controls such as non-targeting gRNA or experimentally verified gRNA can be added in.

After you have the final library of all single-gRNA you want included in your library/screen, you can pair up this user-input sgRNA library to create a library of paired dual-gRNA elements, using excavate-ht pair. We will pair up gRNA about a fixed-point in the MFN2 locus.

excavate-ht pair \
  --input-library final_sgRNA_MFN2_library.csv
  --pairing-method fp\
  --fixed-points-list 11976861 \
  -o paired_MFN2_library