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LihuaJulieZhu / CRISPRseek

Design of target-specific guide RNAs in CRISPR-Cas9, genome-editing systems
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PAM position in gRNAsPlusPAM column #2

Closed FabianDK closed 6 months ago

FabianDK commented 3 years ago

Hello!

I was screening for off-targets using gRNA sequences desgined to target a transgene that normally does not occur in my organism. In the summary.xls, I realized that when a gRNA is not found in the genome at all, then the PAM sequence in the gRNAsPlusPAM column is automatically put to the 3prime end, despite specifying the location to be 5prime.

I also think that for gRNAs not found at all, it would be useful to also show 'perfect match not found'.

Here is my code:

offTargetAnalysis(inputFilePath = gRNAs.input, findPairedgRNAOnly = FALSE, findgRNAs = FALSE, BSgenomeName = BSgenome.Assembly.v1, txdb = txdb.Assembly, orgAnn = org.Assembly.eg.db, outputDir = outpath, overwrite = TRUE, chromToSearch = 'all', max.mismatch = 3, PAM = "TTN", PAM.pattern = "^TTN", PAM.location = '5prime', n.cores.max=6)

Output:

names gRNAsPlusPAM top5OfftargetTotalScore top10OfftargetTotalScore top1Hit.onTarget.MMdistance2PAM topOfftarget1MMdistance2PAM topOfftarget2MMdistance2PAM topOfftarget3MMdistance2PAM topOfftarget4MMdistance2PAM topOfftarget5MMdistance2PAM topOfftarget6MMdistance2PAM topOfftarget7MMdistance2PAM topOfftarget8MMdistance2PAM topOfftarget9MMdistance2PAM topOfftarget10MMdistance2PAM REname
guide1 TTNACCGGGGTGGTGCCCATCCT 2.1 2.1 perfect match not found 2,4,13 3,10,13 4,10,18               BccI BtsCI
guide2 AGCGTGTCCGGCGAGGGCGATTN NA NA NA NA NA NA NA NA NA NA NA NA NA NA
guide3 TTNATCTGCACCACCGGCAAGCT 2 2.2 perfect match not found 1,12,13 3,13,17 5,12,15 5,12,18 2,14,20 6,13,15 13,16,18 12,14,16     AluI Cac8I CviKI-1 MspJI
guide4 CGTCGCCGTCCAGCTCGACCTTN NA NA NA NA NA NA NA NA NA NA NA NA NA NA
guide5 TTNAGGGTCAGCTTGCCGTAGGT 1.1 1.1 perfect match not found 2,3,15 10,13,19 13,14,18               MspJI
LihuaJulieZhu commented 2 years ago

Hi Daniel,

Thank you very much for the feed back! To help me replicate your results, could you please share your input file gRNAs.input?

For your future reference, it would be great if you could post your questions at https://support.bioconductor.org.

Thanks!

Best regards, Julie

hukai916 commented 6 months ago

Hi @FabianDK ,

Sorry for the late response, I can not seem to replicate your issue with the latest version of CRISRPseek. I noticed that you set findgRNAs = FALSE , by doing so, the gRNAs in your gRNAs.input will be directly used for searching off targets. It assumes that the gRNAs.input contain both gRNA and its PAM. If PAM not found in gRNAs.input, it will be added according to PAM and PAM.location. Anyway, thanks for bring this issue up!

--Kai