Hi,

While doing an OT analysis for a specific sequence, I got the following error message:

Error in FUN(X[[i]], ...) : object 'GN' not found

I looked for OTs in mouse genome as following:

seq<-DNAStringSet("GCCCGGAGGCGGGGTGGAGGGGG")
names(seq)<-"test"
offTargetAnalysis(seq,
                         gRNAoutputName=names(seq),
                         format="fasta",
                         BSgenomeName=BSgenome.Mmusculus.UCSC.mm10,
                         findgRNAs = FALSE,
                         gRNA.size = 20,
                         max.mismatch=5,
                         annotatePaired = FALSE,
                         outputDir=out,
                         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 = "NGG",
                         PAM="NGG",
                         PAM.size =3,
                         PAM.location = "3prime",
                         allowed.mismatch.PAM = 0,
                         annotateExon=TRUE)

When doing the same search in human genome using BSgenome.Hsapiens.UCSC.hg38, TxDb.Hsapiens.UCSC.hg38.knownGene and org.Hs.eg.db, I do not have an issue.

Any idea of what it can be?

Thank you in advance for your feedback.

> sessionInfo( )
R version 4.1.0 (2021-05-18)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19043)

Matrix products: default

Dear Julie,

Thank you very much for your fast reply! I will try what you suggest.

Have a great day.

Best, Nicolas

Dear Nicolas,

Thanks for testing CRISPRseek and the feedback!

PAM.pattern should end with a $ sign to indicate that the NGG needs to be in the 3' end.

For your curiosity, the following sequence in Mouse caused the error that you encountered. The PAM.pattern without $ sign will identify the following sequence to be an offtarget. However, there is no activity defined for GN for the CFD scoring algorithm.

GCTTGGAGGA GGAGAGGAGG AGn

Once you set PAM.pattern = "NGG$", it should work. I suggest that you use a less stringent PAM.pattern for research purpose though.

For CRISPRseek Version >= 1.35.2, PAM.pattern will be automatically appended with $ sign if absent and when PAM.location is set to 3prime.

Best,

Julie