Quickstart

The whole pipeline on the bundled C. elegans chrIII test data (tests/test_data/ce11/): build gene models, index the genome, find guides in coding sequence, score them, and pick the best per gene. Each step is one crisprware subcommand; the per-module pages cover the options.

1. Gene models

crisprware preprocess_annotation -g tests/test_data/ce11/chrIII_ce11.ncbiRefSeq.gtf \
  -m metagene consensus longest shortest

Collapses multi-isoform genes into four model GTFs (pick one for the steps below):

chrIII_ce11.ncbiRefSeq/
  chrIII_ce11.ncbiRefSeq_consensus.gtf
  chrIII_ce11.ncbiRefSeq_longestCDS.gtf
  chrIII_ce11.ncbiRefSeq_meta.gtf
  chrIII_ce11.ncbiRefSeq_shortestCDS.gtf

2. Off-target index

crisprware index_genome -f tests/test_data/ce11/chrIII_sequence.fasta -p NGG -l 20

Builds the crispr-ots off-target index into chrIII_sequence_crisprots/ (the .crot mmap database).

3. Generate guides

crisprware generate_guides -f tests/test_data/ce11/chrIII_sequence.fasta \
  -k tests/test_data/ce11/chrIII_ce11.ncbiRefSeq.gtf --feature CDS

Every NGG protospacer whose cut site lands in a CDS -> chrIII_sequence_gRNA/chrIII_sequence_gRNA.bed (312,925 guides):

#chr   start  stop   id,sequence,pam,chromosome,position,sense              context                         strand
chrIII 1482   1502   chrIII:1483:+,GGAATGTACTTCTTCCCAAA,NGG,chrIII,1483,+   TGTTGGAATGTACTTCTTCCCAAAAGGTTC  +

4. Score guides

crisprware score_guides -b chrIII_sequence_gRNA/chrIII_sequence_gRNA.bed \
  -i chrIII_sequence_crisprots/chrIII_sequence_crisprots --tracr Chen2013 --threads 8

Adds an RS3 on-target column and a crispr-ots off-target specificity column (the engine is auto-detected from the index) -> chrIII_sequence_scoredgRNA/...bed:

#chr   start  stop   context                         strand sequence             RS3_score_Chen2013  specificity_chrIII_sequence_crisprots
chrIII 10569  10589  GCTGCCTACATGTACTTTTATTTGAGGGTC  +      CCTACATGTACTTTTATTTG -1.4272             1.0
chrIII 10590  10610  TTGAGGGTCCCCATGATCTTGAAGAGGAGA  +      GGGTCCCCATGATCTTGAAG -0.0695             1.0

RS3 is a z-score (centered at 0); specificity is 0-1 (1.0 = no off-targets).

5. Rank guides

crisprware rank_guides -k chrIII_sequence_scoredgRNA/...bed \
  -t tests/test_data/ce11/chrIII_ce11.ncbiRefSeq.gtf -f CDS \
  -c RS3_score_Chen2013 specificity_chrIII_sequence_crisprots -m 0 0.2 -p 5 65 \
  -r RS3_score_Chen2013 --output_all

Keeps guides with RS3 >= 0, specificity >= 0.2, in the 5th-65th CDS percentile, ranks by RS3, and returns the picks per gene -> ..._rankedgRNA/...bed:

#chr   start   stop    strand sequence             RS3_score_Chen2013  specificity_...  target_id  RS3_..._normalized  combined_weighted
chrIII 574162  574182  +      TAAGCTTAGTACTTATGAGG 0.9041              1.0              B0353.1    0.8819              0.8819