Find prime editing spacers

Find prime editing spacers around target ranges

find_primespacers(
  gr,
  bsgenome,
  edits = get_plus_seq(bsgenome, gr),
  nprimer = 13,
  nrt = 16,
  ontargetmethod = c("Doench2014", "Doench2016")[1],
  offtargetmethod = c("bowtie", "pdict")[1],
  mismatches = 0,
  nickmatches = 2,
  indexedgenomesdir = INDEXEDGENOMESDIR,
  outdir = OUTDIR,
  verbose = TRUE,
  plot = TRUE,
  ...
)

Arguments

gr

GRanges-class
bsgenome

BSgenome-class
edits

character vector: desired edits on '+' strand. If named, names should be identical to those of gr
nprimer

n primer nucleotides (default 13, max 17)
nrt

n rev transcr nucleotides (default 16, recomm. 10-16)
ontargetmethod

'Doench2014' or 'Doench2016': on-target scoring method
offtargetmethod

'bowtie' or 'pdict'
mismatches

no of primespacer mismatches (default 0, to suppress offtarget analysis: -1)
nickmatches

no of nickspacer offtarget mismatches (default 2, to suppresses offtarget analysis: -1)
indexedgenomesdir

directory with indexed genomes (as created by index_genome)
outdir

directory whre offtarget analysis output is written
verbose

TRUE (default) or FALSE
plot

TRUE (default) or FALSE
...

passed to plot_intervals

Value

GRanges-class with prime editing spacer ranges and following mcols: * crisprspacer: N20 spacers * crisprpam: NGG PAMs * crisprprimer: primer (on PAM strand) * crisprtranscript: reverse transcript (on PAM strand) * crisprextension: 3' extension of gRNA contains: reverse transcription template + primer binding site sequence can be found on non-PAM strand * crisprextrange: genomic range of crispr extension * Doench2016|4: on-target efficiency scores * off0, off1, off2: number of offtargets with 0, 1, 2 mismatches * off: total number of offtargets: off = off0 + off1 + ... * nickrange: nickspacer range * nickspacer: nickspacer sequence * nickDoench2016|4: nickspacer Doench scores * nickoff: nickspacer offtarget counts

Details

Below the architecture of a prime editing site. Edits can be performed anywhere in the revtranscript area.

spacer pam --------------------=== primer revtranscript -------------================ 1..............17....GG.......... .....................CC.......... ----------extension----------

See also

find_spacers to find standard crispr sites

Examples

# Find PE spacers for 4 clinically relevant loci (Anzalone et al, 2019)
    bsgenome <- BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38
    gr <- char_to_granges(c(
        PRNP = 'chr20:4699600:+',             # snp: prion disease
        HBB  = 'chr11:5227002:-',             # snp: sickle cell anemia
        HEXA = 'chr15:72346580-72346583:-',   # del: tay sachs disease
        CFTR = 'chr7:117559593-117559595:+'), # ins: cystic fibrosis
        bsgenome)
    spacers <- find_primespacers(gr, bsgenome)
#> Find primespacers for 4 targets
#> 	Found 10 primespacers
#> 	Count offtargets with max 0 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 53 hits with max 1 mismatch(es)
#> 				Retain 20 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 20 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 20 after removing NGG 'G2' mismatches
#> 				Retain 11 after removing more than 0 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       9 are off0 free
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Find nickspacers
#> Find spacers in 10 targets
#> 	Found 32 spacers
#> 	Count offtargets with max 2 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 91 hits with max 2 mismatch(es)
#> 				Retain 37 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 36 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 33 after removing NGG 'G2' mismatches
#> 				Retain 33 after removing more than 2 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       30 are off0 free
#> 		       32 are off1 free
#> 		       24 are off2 free
#> 	Filter for best offtarget counts
#> 		Retain 32 with best off0 counts per pename
#> 		Retain 32 with best off1 counts per pename
#> 		Retain 24 with best off2 counts per pename
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Warning: Removed 34 rows containing missing values (geom_point).
#> Warning: Removed 34 rows containing missing values (geom_point).
    spacers <- find_spacers(extend_for_pe(gr), bsgenome, complement = FALSE)
#> Find spacers in 8 targets
#> 	Found 10 spacers
#> 	Count offtargets with max 2 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 140 hits with max 2 mismatch(es)
#> 				Retain 80 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 77 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 71 after removing NGG 'G2' mismatches
#> 				Retain 71 after removing more than 2 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       9 are off0 free
#> 		       9 are off1 free
#> 		       2 are off2 free
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014

# Edit PRNP locus for resistance against prion disease (Anzalone et al, 2019)
    bsgenome <- BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38
    gr <- char_to_granges(c(PRNP = 'chr20:4699600:+'), bsgenome)
    find_primespacers(gr, bsgenome)
#> Find primespacers for 1 targets
#> 	Found 4 primespacers
#> 	Count offtargets with max 0 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 25 hits with max 1 mismatch(es)
#> 				Retain 13 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 13 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 13 after removing NGG 'G2' mismatches
#> 				Retain 4 after removing more than 0 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       4 are off0 free
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Find nickspacers
#> Find spacers in 4 targets
#> 	Found 12 spacers
#> 	Count offtargets with max 2 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 13 hits with max 2 mismatch(es)
#> 				Retain 4 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 4 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 3 after removing NGG 'G2' mismatches
#> 				Retain 3 after removing more than 2 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       12 are off0 free
#> 		       12 are off1 free
#> 		       12 are off2 free
#> 	Filter for best offtarget counts
#> 		Retain 12 with best off0 counts per pename
#> 		Retain 12 with best off1 counts per pename
#> 		Retain 12 with best off2 counts per pename
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Warning: Removed 16 rows containing missing values (geom_point).
#> Warning: Removed 16 rows containing missing values (geom_point).
#> GRanges object with 4 ranges and 21 metadata columns:
#>          seqnames          ranges strand |  targetname targetstart targetend
#>             <Rle>       <IRanges>  <Rle> | <character>   <integer> <integer>
#>   PRNP_1    chr20 4699568-4699587      + |        PRNP     4699600   4699600
#>   PRNP_2    chr20 4699569-4699588      + |        PRNP     4699600   4699600
#>   PRNP_3    chr20 4699575-4699594      + |        PRNP     4699600   4699600
#>   PRNP_4    chr20 4699578-4699597      + |        PRNP     4699600   4699600
#>           crisprname         crisprspacer   crisprpam  crisprprimer
#>          <character>          <character> <character>   <character>
#>   PRNP_1      PRNP_1 AGCAGCTGGGGCAGTGGTGG         GGG GCTGGGGCAGTGG
#>   PRNP_2      PRNP_2 GCAGCTGGGGCAGTGGTGGG         GGG CTGGGGCAGTGGT
#>   PRNP_3      PRNP_3 GGGGCAGTGGTGGGGGGCCT         TGG CAGTGGTGGGGGG
#>   PRNP_4      PRNP_4 GCAGTGGTGGGGGGCCTTGG         CGG TGGTGGGGGGCCT
#>          crisprtranscript               crisprextension          crisprextrange
#>               <character>                   <character>             <character>
#>   PRNP_1 TGGGGGGCCTTGGCGG CCGCCAAGGCCCCCCACCACTGCCCCAGC chr20:4699572-4699600:-
#>   PRNP_2 GGGGGGCCTTGGCGGC GCCGCCAAGGCCCCCCACCACTGCCCCAG chr20:4699573-4699601:-
#>   PRNP_3 CCTTGGCGGCTACATG CATGTAGCCGCCAAGGCCCCCCACCACTG chr20:4699579-4699607:-
#>   PRNP_4 TGGCGGCTACATGCTG CAGCATGTAGCCGCCAAGGCCCCCCACCA chr20:4699582-4699610:-
#>                off      off0 Doench2014
#>          <numeric> <numeric>  <numeric>
#>   PRNP_1         0         0 0.00994899
#>   PRNP_2         0         0 0.04496627
#>   PRNP_3         0         0 0.00996254
#>   PRNP_4         0         0 0.20935863
#>                                                                        nickrange
#>                                                                      <character>
#>   PRNP_1 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_2 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_3 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_4 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>                                                              nickspacer
#>                                                             <character>
#>   PRNP_1 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_2 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_3 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_4 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>              nickpam     nickoff    nickoff0    nickoff1    nickoff2
#>          <character> <character> <character> <character> <character>
#>   PRNP_1 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_2 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_3 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_4 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>          nickDoench2014
#>             <character>
#>   PRNP_1 0.14;0.32;0.52
#>   PRNP_2 0.14;0.32;0.52
#>   PRNP_3 0.14;0.32;0.52
#>   PRNP_4 0.14;0.32;0.52
#>   -------
#>   seqinfo: 595 sequences (1 circular) from hg38 genome
    find_primespacers(gr, bsgenome, edits = 'T')
#> Find primespacers for 1 targets
#> 	Found 4 primespacers
#> 	Count offtargets with max 0 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 25 hits with max 1 mismatch(es)
#> 				Retain 13 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 13 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 13 after removing NGG 'G2' mismatches
#> 				Retain 4 after removing more than 0 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       4 are off0 free
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Find nickspacers
#> Find spacers in 4 targets
#> 	Found 12 spacers
#> 	Count offtargets with max 2 mismatches using bowtie
#> 		Count genome (mis)matches
#> 			Expand iupac ambiguities in pam
#> 			Write reads to ~/multicrisprout/spacers.fa
#> 			Map reads: ~/multicrisprout/spacers/spacers_to_BSgenome.Hsapiens.UCSC.hg38.txt
#> 			Load results
#> 				Read 13 hits with max 2 mismatch(es)
#> 				Retain 4 after removing NGG 'N' mismatches (avoid double counting expanded pams)
#> 				Retain 4 after removing NGG 'G1' mismatches(except for NGG -> NAG, which is allowed)
#> 				Retain 3 after removing NGG 'G2' mismatches
#> 				Retain 3 after removing more than 2 mismatches
#> 			Count matches
#> 		Count off-targets
#> 		       12 are off0 free
#> 		       12 are off1 free
#> 		       12 are off2 free
#> 	Filter for best offtarget counts
#> 		Retain 12 with best off0 counts per pename
#> 		Retain 12 with best off1 counts per pename
#> 		Retain 12 with best off2 counts per pename
#> 	Score ontargets
#> 		Add (4-23-3) contextseqs
#> 		Score contextseqs with Doench2014
#> Warning: Removed 16 rows containing missing values (geom_point).
#> Warning: Removed 16 rows containing missing values (geom_point).
#> GRanges object with 4 ranges and 21 metadata columns:
#>          seqnames          ranges strand |  targetname targetstart targetend
#>             <Rle>       <IRanges>  <Rle> | <character>   <integer> <integer>
#>   PRNP_1    chr20 4699568-4699587      + |        PRNP     4699600   4699600
#>   PRNP_2    chr20 4699569-4699588      + |        PRNP     4699600   4699600
#>   PRNP_3    chr20 4699575-4699594      + |        PRNP     4699600   4699600
#>   PRNP_4    chr20 4699578-4699597      + |        PRNP     4699600   4699600
#>           crisprname         crisprspacer   crisprpam  crisprprimer
#>          <character>          <character> <character>   <character>
#>   PRNP_1      PRNP_1 AGCAGCTGGGGCAGTGGTGG         GGG GCTGGGGCAGTGG
#>   PRNP_2      PRNP_2 GCAGCTGGGGCAGTGGTGGG         GGG CTGGGGCAGTGGT
#>   PRNP_3      PRNP_3 GGGGCAGTGGTGGGGGGCCT         TGG CAGTGGTGGGGGG
#>   PRNP_4      PRNP_4 GCAGTGGTGGGGGGCCTTGG         CGG TGGTGGGGGGCCT
#>          crisprtranscript               crisprextension          crisprextrange
#>               <character>                   <character>             <character>
#>   PRNP_1 TGGGGGGCCTTGGCGT ACGCCAAGGCCCCCCACCACTGCCCCAGC chr20:4699572-4699600:-
#>   PRNP_2 GGGGGGCCTTGGCGTC GACGCCAAGGCCCCCCACCACTGCCCCAG chr20:4699573-4699601:-
#>   PRNP_3 CCTTGGCGTCTACATG CATGTAGACGCCAAGGCCCCCCACCACTG chr20:4699579-4699607:-
#>   PRNP_4 TGGCGTCTACATGCTG CAGCATGTAGACGCCAAGGCCCCCCACCA chr20:4699582-4699610:-
#>                off      off0 Doench2014
#>          <numeric> <numeric>  <numeric>
#>   PRNP_1         0         0 0.00994899
#>   PRNP_2         0         0 0.04496627
#>   PRNP_3         0         0 0.00996254
#>   PRNP_4         0         0 0.20935863
#>                                                                        nickrange
#>                                                                      <character>
#>   PRNP_1 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_2 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_3 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>   PRNP_4 chr20:4699632-4699651:-;chr20:4699633-4699652:-;chr20:4699664-4699683:-
#>                                                              nickspacer
#>                                                             <character>
#>   PRNP_1 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_2 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_3 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>   PRNP_4 TCACTGCCGAAATGTATGAT;GTCACTGCCGAAATGTATGA;GCATGTTTTCACGATAGTAA
#>              nickpam     nickoff    nickoff0    nickoff1    nickoff2
#>          <character> <character> <character> <character> <character>
#>   PRNP_1 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_2 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_3 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>   PRNP_4 GGG;TGG;CGG       0;0;0       0;0;0       0;0;0       0;0;0
#>          nickDoench2014
#>             <character>
#>   PRNP_1 0.14;0.32;0.52
#>   PRNP_2 0.14;0.32;0.52
#>   PRNP_3 0.14;0.32;0.52
#>   PRNP_4 0.14;0.32;0.52
#>   -------
#>   seqinfo: 595 sequences (1 circular) from hg38 genome