Hello,

I have a question about “gRNAefficacy” in “OfftargetAnalysis.xls” file. I used to use this code below and was getting “gRNAefficacy” column but recently I noticed this column is not there (or contains NA). Guide that I'm testing should not have a perfect match in the genome (it is a negative control). Am I assuming correctly that if your score < 1, “gRNAefficacy” will be “NA”? Previously, even if guide didn't have a perfect match in the genome “gRNAefficacy” column was still calculated. Do I need to include another argument in offTargetAnalysis()?

Thanks, Dawid

offTargetAnalysis(inputFilePath=inputFilePath,
                  gRNAoutputName=outputDir, #enter a name for the gRNA ouput file name when DNAStringSet instead of file path provided
                  outputDir = outputDir,
                  REpatternFile = REpatternFile,
                  scoring.method = "CFDscore",
                  min.score = 0.000001,  
                  format = "fasta",
                  findgRNAs = FALSE, # important for testing guides to set FALSE
                  findgRNAsWithREcutOnly = FALSE, # if FALSE not restr. enzymes
                  findPairedgRNAOnly = FALSE, # paired only turn to FALSE if only WT Cas9 not Nickase
                  gRNA.name.prefix = "g.",                 
                  orgAnn = orgAnn,
                  BSgenomeName = BSgenomeName,
                  txdb = txdb,
                  annotateExon = TRUE,
                  chromToSearch= "all", # change here for all to look at all chromosomes
                  min.gap = 0, max.gap = 20,
                  max.mismatch = 4,
                  topN = 1000,
                  topN.OfftargetTotalScore= 10, # 10 top Offtarget will be calculated 
                  fetchSequence = TRUE, upstream = 250, downstream = 250,
                  overlap.gRNA.positions = c(17, 18), 
                  PAM.size = 3,
                  PAM = "NGG",
                  gRNA.size = 20,
                  overwrite = TRUE)

> sessionInfo() R version 4.0.2 (2020-06-22) Platform: x86_64-apple-darwin17.0 (64-bit) Running under: macOS Catalina 10.15.6

Matrix products: default BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib

locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

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

other attached packages:  [1] RankAggreg_0.6.6                         plyr_1.8.6                                forcats_0.5.0                [4] stringr_1.4.0                             dplyr_1.0.2              purrr_0.3.4                                [7] readr_1.3.1             tidyr_1.1.1                               tibble_3.0.3                 [10] tidyverse_1.3.0                           org.Hs.eg.db_3.11.4     TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2   [13] BSgenome.Hsapiens.UCSC.hg19_1.4.3         org.Mm.eg.db_3.11.4          TxDb.Mmusculus.UCSC.mm10.knownGene_3.10.0 [16] GenomicFeatures_1.40.1  AnnotationDbi_1.50.3                      Biobase_2.48.0               [19] BSgenome.Mmusculus.UCSC.mm10_1.4.0        Hmisc_4.4-1             ggplot2_3.3.2                             [22] Formula_1.2-3           survival_3.2-3                            lattice_0.20-41              [25] readxl_1.3.1                              seqinr_3.6-1            CRISPRseek_1.28.0                         [28] BSgenome_1.56.0         rtracklayer_1.48.0                        Biostrings_2.56.0            [31] XVector_0.28.0                            GenomicRanges_1.40.0    GenomeInfoDb_1.24.2                       [34] IRanges_2.22.2          S4Vectors_0.26.1                          BiocGenerics_0.34.0

Hi Dawid,

Your assumption is correct that if your score < 1, “gRNAefficacy” will be “NA". Previously, even if guide didn't have a perfect match in the genome “gRNAefficacy” column was still calculated.

According to the feedback I received, if the score < 1, "gRNAefficacy" is not applicable. To speed up the analysis, I updated the code to set “gRNAefficacy” to “NA" if your score < 1.

Do you think it is important to add another argument in offTargetAnalysis to give users the option to have it calculated or not?

Thanks!

Best regards,

Julie

Hi Dawid,

Thanks for sharing your use cases with gRNAefficancy for offtarget sites!

I will add a parameter calculategRNAefficacyForOfftargets, default to TRUE.

Meanwhile, you can use the function calculategRNAEfficiency to get gRNAefficacy for the offtargets. Please set upstream to 4 and downstream to 3 in offTargetAnalysis call and save the output to a variable.

Here is an example.

library(CRISPRseek) library("BSgenome.Hsapiens.UCSC.hg19") library(TxDb.Hsapiens.UCSC.hg19.knownGene) library(org.Hs.eg.db) outputDir <- getwd() inputFilePath <- system.file("extdata", "inputseq.fa", package = "CRISPRseek")

results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, 
    findPairedgRNAOnly = FALSE, 
        annotatePaired = FALSE,
        BSgenomeName = Hsapiens, chromToSearch = "chrX",
        txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, 
    orgAnn = org.Hs.egSYMBOL, max.mismatch = 1, upstream = 4, downstream = 3,
        outputDir = outputDir, overwrite = TRUE)


featureWeightMatrixFile <- system.file("extdata", "DoenchNBT2014.csv", 
    package = "CRISPRseek")
featureWeightMatrix <- read.csv(featureWeightMatrixFile, header=TRUE)
gRNAefficacy <- calculategRNAEfficiency(results$offtarget$flankSequence, baseBeforegRNA = 4, 
    featureWeightMatrix, gRNA.size = 20)

Hi Dawid,

I have added a parameter called calculategRNAefficacyForOfftargets in offTargetAnalysis. By default, it will output gRNAefficacy for ontargets as well as offtargets.

The updated function is available in CRISPRseek_1.29.1.

Please let me know if it works as expected.

Thanks for your feedback and sharing your use cases! To benefit others, I have added this link to the help menu of offTargetAnalysis function.

Best regards,

Julie