Dear Emiliano,

You will need to modify gRNA.size to 18 and weights to have 18 numbers instead of 20 numbers to the call .

Here is the modified function call

offTargetAnalysis(gRNAFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, findgRNAs = FALSE, BSgenomeName = Mmusculus, chromToSearch = 'chrY', txdb = TxDb.Mmusculus.UCSC.mm10.knownGene, orgAnn = org.Mm.egSYMBOL, max.mismatch = 3, outputDir = outputDir, overwrite = TRUE,

gRNA.size = 18,

weights = c(0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445,
        0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583)

)

Best regards,

Julie

From: Emiliano Ricci <ricci.emiliano@gmail.com>
Date: Thursday, October 1, 2015 8:30 AM
To: Lihua Julie Zhu <julie.zhu@umassmed.edu>
Subject: Using CRISPRseek with shorter gRNAs

Dear Julie,

I am a former postdoc in Melissa Moore laboratory (at UMASS) and now an assistant professor in France. I have been using CRISPRseek for the past few months and have been very happy with it. 

I have recently started to use shorter gRNAs (18nt) because they have been shown to decrease off-target effects as a mismatch with the target genomic DNA is more penalising than with a longer gRNA. I therefore wanted to compare the off-target predictions for a 20nt and a 18nt gRNA using CRISPRseek. However, whenever I have a shorter gRNA, it appears as CRISPRseek is able to scan for off-targets but then fails when annotating the file. I get the following error:

Validating input ...

>>> Finding all hits in sequence chrY ...

>>> DONE searching

Error in scan(file, what, nmax, sep, dec, quote, skip, nlines, na.strings,  : 

  line 5 did not have 28 elements

This is the command that I used:

setwd("/Users/adminer/Box Sync/TEV_Lab/Projets/Macrophage_Translation_Control/CRISPRseek/METTL3_METTL14_Introns")

gRNAFilePath <- ('/Users/adminer/Box Sync/TEV_Lab/Projets/Macrophage_Translation_Control/CRISPRseek/METTL3_METTL14_Introns/target_intronsallgRNAs.fa')

outputDir <- getwd()

REpatternFile <- system.file('extdata', 'NEBenzymes.fa', package = 'CRISPRseek')

results <- offTargetAnalysis(gRNAFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile, findPairedgRNAOnly = FALSE, findgRNAs = FALSE, BSgenomeName = Mmusculus, chromToSearch = 'chrY', txdb = TxDb.Mmusculus.UCSC.mm10.knownGene, orgAnn = org.Mm.egSYMBOL, max.mismatch = 3, outputDir = outputDir, overwrite = TRUE)

Is it possible to use the off-target prediction function with external gRNA sequences that are shorter than 20nt? Is there an argument that I can add to the command to tell the software the gRNA is shorter?

Thank you very much for your attention.

Best regards, 

Emiliano Ricci.