Hi,

I am currently searching for off target(s) for specific gRNAs with a PAM site in 5' using the following function where input 1 is my gRNA in fasta format.

offTargetAnalysis(
                    input1,
                    format="fasta",
                    BSgenomeName=BSgenome.Mmusculus.UCSC.mm10,
                    findgRNAs = FALSE,
                    gRNA.size = 23,
                    max.mismatch=4,
                    annotatePaired = FALSE,
                    outputDir=outDir,
                    enable.multicore = TRUE,
                    n.cores.max = 6,
                    scoring.method="CFDscore",
                    txdb = TxDb.Mmusculus.UCSC.mm10.knownGene,
                    orgAnn = org.Mm.eg.db,
                    predictIndelFreq.onTargetOnly=FALSE,
                    calculategRNAefficacyForOfftargets=FALSE,
                    useScore=FALSE,
                    PAM.pattern = "^YTTN",
                    PAM="CTTG",
                    PAM.size = 4,
                    PAM.location = "5prime",
                    allowed.mismatch.PAM = 1)

I am not expecting a match and got none but in the output document the column gRNAsPlusPAM is displaying the gRNA with the PAM site in 3' instead of 5'. I assume then that the search is done with the same configuration. Any idea on what I am doing wrong here?

Thanks in advance

sessionInfo( )
R version 4.0.3 (2020-10-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

locale:
[1] LC_COLLATE=English_Denmark.1252 
[2] LC_CTYPE=English_Denmark.1252   
[3] LC_MONETARY=English_Denmark.1252
[4] LC_NUMERIC=C                    
[5] LC_TIME=English_Denmark.1252    

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices
[6] utils     datasets  methods   base     

other attached packages:
 [1] readxl_1.3.1                             
 [2] forcats_0.5.1                            
 [3] stringr_1.4.0                            
 [4] dplyr_1.0.5                              
 [5] purrr_0.3.4                              
 [6] readr_1.4.0                              
 [7] tidyr_1.1.3                              
 [8] tibble_3.1.0                             
 [9] ggplot2_3.3.3                            
[10] tidyverse_1.3.0                          
[11] xlsx_0.6.5                               
[12] seqinr_4.2-5                             
[13] org.Mm.eg.db_3.12.0                      
[14] TxDb.Mmusculus.UCSC.mm10.knownGene_3.10.0
[15] GenomicFeatures_1.42.3                   
[16] AnnotationDbi_1.52.0                     
[17] Biobase_2.50.0                           
[18] BSgenome.Mmusculus.UCSC.mm10_1.4.0       
[19] BSgenome_1.58.0                          
[20] rtracklayer_1.49.5                       
[21] GenomicRanges_1.42.0                     
[22] GenomeInfoDb_1.26.4                      
[23] CRISPRseek_1.30.1                        
[24] Biostrings_2.58.0                        
[25] XVector_0.30.0                           
[26] IRanges_2.24.1                           
[27] S4Vectors_0.28.1                         
[28] BiocGenerics_0.36.0

Dear Nicolas,

Thanks for the example in which there is no match found in the genome! I have modified the code to handle this special case with PAM.location = "5prime".

The updated version is available as Version: 1.31.2. It will take a couple of days to show up at http://bioconductor.org/packages/devel/bioc/html/CRISPRseek.html

if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")

The following initializes usage of Bioc devel

BiocManager::install(version='devel')

BiocManager::install("CRISPRseek")

In addition, I would like to point out that the predicted offtarget scores and efficacies are not reliable when you use CRISPR systems other than spCas9 since the predictive models were based on data from spCas9. We will be happy to try to add additional prediction algorithms per your recommendation.

To speed up the test runs, I recommend set max.mismatch = 0 or 1 or use chromToSearch to strict the search to a subset of the chromosomes.

Thanks again for providing us this special example which can now be handled properly by CRISPRseek.

Best regards,

Julie

Dear Julie,

Tank you very much for your very fast answer and help.

Best regards, Nicolas