Index and guides

Two preparatory builds: the off-target index every guide is scanned against, and the guide set itself.

1. Off-target index (PAM `TTTN`)

crisprware index_genome \
  -f $PROJ/hg38_primary_chr.fna \
  --pam TTTN \
  -l 23 \
  --pam_5_prime \
  --bin_width 14 \
  -o $PROJ

Flag	Meaning
`--pam TTTN`	index PAM. `TTTN`, not `TTTV`, so weak-PAM (`TTTT`) off-targets are enumerable.
`-l 23`	23-nt protospacer length (Cas12a).
`--pam_5_prime`	PAM sits 5’ of the spacer (Cas12a geometry).
`--bin_width 14`	index bin width; 14 is the measured throughput optimum that still fits a 24 GB GPU.

Output: hg38_primary_chr_crisprots/ (the index, ~7 GB for hg38). Build is single-threaded-ish and memory-light (~19 GB RAM, ~9-22 min for a human genome).

2. Enumerate `TTTV` protospacers

crisprware generate_guides \
  -f $PROJ/hg38_primary_chr.fna \
  --pam TTTV \
  --pam_5_prime \
  -5 19 -3 23 \
  -l 23 \
  -w 4 3 \
  --threads 16 \
  -o $PROJ

Flag	Meaning
`--pam TTTV`	guide PAM, the strong canonical set (`V = A/C/G`, excludes the weak `TTTT`).
`-5 19 -3 23`, `-l 23`	protospacer extent relative to the PAM.
`-w 4 3`	context window: 4 nt up + 4 PAM + 23 spacer + 3 down = a 34-nt window, exactly what the DeepCpf1 / EnPAM-GB / enseq-DeepCpf1 models expect.
`--threads 16`	window-parallel engine; ~8x faster genome-wide.

Output: hg38_primary_chr_gRNA/hg38_primary_chr_gRNA.bed, one row per guide.

guides: 135,867,611

Tip

The index is TTTN but the guides are TTTV. That is deliberate (see the overview): we score strong-PAM guides against an index that can still find their weak-PAM off-targets.