Abstract gRNA

To encapsulate motifs that we search for on the genome, we can use abstract definition of gRNA/off-target, which is defined in Motif data structure.

CHOPOFF.Motif — Type

Motif(
    alias::String, 
    fwdmotif::String, 
    fwdpam::String, 
    forward_strand::Bool = true, 
    reverse_strand::Bool = true, 
    distance::Int = 4, 
    extends5::Bool = true,
    ambig_max::Int = 5)

Motif(alias::String)

Motif defines what we search on the genome, what can be identified as an off-target.

Arguments

alias - alias of the motif for easier identification e.g. Cas9

fwdmotif - Motif that indicates where is PAM inside fwdpam. For example for Cas9 it is 20*N + XXX: NNNNNNNNNNNNNNNNNNNNXXX

fwdpam - Motif in 5'-3' that will be matched on the reference (without the X). For example for Cas9 it is 20*X + NGG: XXXXXXXXXXXXXXXXXXXXNGG

forward - If false will not match to the forward reference strand.

reverse - If false will not match to the reverse reference strand.

distance - How many extra nucleotides are needed for a search? This will indicate within what distance we can search for off-targets. When we don't have those bases we use DNA_Gap.

extend5 - Defines how off-targets will be aligned to the guides and where extra nucleotides will be added for alignment within distance. Whether to extend in the 5' and 3' direction. Cas9 is extend5 = true.

ambig_max- How many ambiguous bases are allowed in the pattern?

Example for Cas9 where we want to search for off-targets within distance of 4:
  alias:    Cas9
  fwdmotif: NNNNNNNNNNNNNNNNNNNNXXX
  fwdpam:   XXXXXXXXXXXXXXXXXXXXNGG
  forward:  true
  reverse:  true
  distance: 4
  extend5:  true
  ambig_max:5

Alignments will be performed from opposite to the extension direction (which is defined by extend5).

Examples

julia> Motif("Cas9")
Alias: Cas9
Maximum search distance: 3
Number of allowed ambigous bp: 0
20N-NGG

julia> Motif("test name", "NNNNNNNNNNNNNNNNNNNNXXX", "XXXXXXXXXXXXXXXXXXXXNGG", true, true, 3, true, 5)
Alias: test name
Maximum search distance: 3
Number of allowed ambigous bp: 5
20N-NGG

CHOPOFF.length_noPAM — Function

length_noPAM(motif::Motif)

Calculate what is the length of the motif, without extension, and without PAM. Effectively, size of the gRNA.

Examples

julia> length_noPAM(Motif("Cas9"))
20

CHOPOFF.setdist — Function

setdist(motif::Motif, distance::Int)

Set the distance (maximum value of allowed mismatches, deletion, insertions) that are allowed during alignment.

Examples

julia> setdist(Motif("Cas9"), 15)
Alias: Cas9
Maximum search distance: 15
Number of allowed ambigous bp: 0
20N-NGG

CHOPOFF.setambig — Function

setambig(motif::Motif, ambig::Int)

Set the ambiguity (how many ambiguous bases are allowed, not counting PAM, not counting extension) level for motif.

Examples

julia> setambig(Motif("Cas9"), 15)
Alias: Cas9
Maximum search distance: 3
Number of allowed ambigous bp: 15
20N-NGG