Cas9 tools

Find Cas9 binding sites

Find Cas9 binding sites for HeliCas-FISH. Outputs input_intervals.fa which contains sequences between binding sites for subsequent probe design, and input_sgRNAs.fa contains guide RNA sequences.

usage: cas9BDFinder.py [-h] [-r] [-g1 G1] [-g2 G2] [-d1 D1] [-d2 D2] [-clusterMinSize CLUSTERMINSIZE] sequence.fasta N

Find Cas9 binding sites for HeliCas-FISH. Run tests if no input is provided.

positional arguments:
  sequence.fasta        target genomic sequence(s).
  N                     max number of binding sites needed.

optional arguments:
  -h, --help            show this help message and exit
  -r                    search the reverse complementary strand.
  -g1 G1                min GC content of protospacers. Default to 0.35.
  -g2 G2                max GC content of protospacers. Default to 0.75.
  -d1 D1                min distance between binding sites. Default to 50.
  -d2 D2                max distance between binding sites. Default to 200.
  -clusterMinSize CLUSTERMINSIZE
                        minimum number of Cas9 binding sites in one cluster.
                        Default to 1.

Design FISH probes with OligoArray

Run OligoArray on regions between identified Cas9 binding sites.

Finalize Cas9 sgRNA sequences

Filtered the FISH probes for good quality and discard Cas9 sites flanking bad probes.

usage: oligoFilter.py [-h] oligo.txt sgRNA.fasta interval.fasta max_nonspecific num_probes

Throw out bad sgRNAs and FISH probes based on OligoArray output. Outputs
oligos_filtered.tsv, sgRNAs_filtered.fasta, and intervals_filtered.fasta

positional arguments:
  oligo.txt        Output from OligoArray.
  sgRNA.fasta      sgRNA candidates from cas9BDFinder.py
  interval.fasta   Sequence between sgRNAs that belong to one cluster from cas9BDFinder.py
  max_nonspecific  Maximum number of nonspecific sites allowed for one FISH probe
  num_probes       Maximum number of probes needed for one region

optional arguments:
  -h, --help       show this help message and exit