Help with using CRISPRseeker
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@sharvari-gujja-6614
Last seen 14 months ago
United States

Hi Julie,

I am writing to seek your expertise in using CRISPRseeker tool to evaluate the accuracy and efficiency of CRISPR experiment.

Particularly, I am looking at "Scenario 5. Target and off-target analysis for gRNAs input by user" in the manual: http://bioconductor.org/packages/release/bioc/manuals/CRISPRseek/man/CRISPRseek.pdf

To briefly describe the experimental design, I have three biological replicates of target gene KO (using CRISPR/Cas) and control (non-target) samples from human cancer cell line. Running alignment and quantification using STAR results in very low counts for target gene in control samples, and higher counts in KO samples. I would really appreciate if you could provide some advice on leveraging CRISPRseeker tool to address this situation. Also, on how to interpret metric value for gRNAefficacy - does low value indicate low on-target rate?

I really appreciate all the help. Thanks
CRISPR • 2.2k views
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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Hi Sharvari,

Yes, you can evaluate the efficiency/efficacy and specificity (offtargets) of your gRNA by following Scenario 5 of the user guide. Please set scoring.method = "CFDscore" for obtaining the cutting frequency determination (CFD) score for offtargets, as described in the section of Scenario 10. To obtain the gRNA efficacy using the rule set 2 published in 2016 by Root lab, please set rule.set = "RootRuleSet22016", as described in the section of Scenario 7. To search for offtargets in the whole genome, please set chromToSearch = "all", and max.mismatch = 3. Please note that the larger the max.mismatch you set, the longer time it takes to run the search. To find more information on other parameters, please type help(offTargetAnalysis) in a R session.

Your interpretation of the value of gRNA efficacy is correct, i.e., the lower the value, the lower the on-target rate.

Best regards,

Julie

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Hi Julie,

Thank you for the reply. I was wondering if the input for "REpatternFile" should be the same as used in the manual? Or, is it specific to individual experiment?

Thanks Sharvari

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Hi Julie,

Thank you for the reply. I was wondering if the input for "REpatternFile" should be the same as used in the manual? Or, is it specific to individual experiment?

Thanks Sharvari

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Hi Julie,

Thank you for the reply. I was wondering if the input for "REpatternFile" should be the same as used in the manual? Or, is it specific to individual experiment?

Thanks Sharvari

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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Hi Sharvari, It is the same as used in the manual. Best regards, Julie
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Hi Julie,

Thanks again. I am getting an error trying to run the function 'offTargetAnalysis'. Can you please help.

results <- offTargetAnalysis(inputFilePath = gRNAFilePath,
                             findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile,
                             scoring.method = "CFDscore",
                             rule.set = "Root_RuleSet2_2016",
                             findPairedgRNAOnly = FALSE, findgRNAs = FALSE,
                             BSgenomeName = Hsapiens, chromToSearch = 'all',
                             txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,
                             orgAnn = org.Hs.egSYMBOL,
                             max.mismatch = 1, outputDir = outputDir, overwrite = TRUE)

DONE searching Building feature vectors for scoring ... Calculating scores ... Annotating, filtering and generating reports ... Python 3.7.1 Error in read.table("pythonVersion.txt", sep = "", header = FALSE) : no lines available in input Calls: offTargetAnalysis ... filterOffTarget -> calculategRNAEfficiency2 -> grep -> read.table Execution halted

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Sharvari,

Please try the following script.

results <- offTargetAnalysis(inputFilePath = gRNAFilePath,

                         findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile,

                         scoring.method = "CFDscore",

                         findPairedgRNAOnly = FALSE, findgRNAs = FALSE,

                         BSgenomeName = Hsapiens, chromToSearch = 'all',

                         txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,

                         orgAnn = org.Hs.egSYMBOL,

                         max.mismatch = 1, outputDir = outputDir, overwrite = TRUE)

If you can run the above script successfully, please try to follow the Scenario 7 at https://www.bioconductor.org/packages/release/bioc/vignettes/CRISPRseek/inst/doc/CRISPRseek.pdf Specifically, in order to use Rule set 2, first install python 2.7, then install the python packages: scikit-learn 0.16.1, pickle, pan- das, numpy nd scipy. In a R session, type the following script to use python 2.7 since Rule set 2 is implemented in python 2.7.

Sys.setenv(PATH = paste("~/anaconda2/bin", Sys.getenv("PATH"), sep=":"))

system("python --version")

Python 2.7.15 :: Anaconda, Inc.

Now, the following script should run fine.

results <- offTargetAnalysis(inputFilePath = gRNAFilePath,

                         findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile,

                         scoring.method = "CFDscore",

                         rule.set = "Root_RuleSet2_2016",

                         findPairedgRNAOnly = FALSE, findgRNAs = FALSE,

                         BSgenomeName = Hsapiens, chromToSearch = 'all',

                         txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,

                         orgAnn = org.Hs.egSYMBOL,

                         max.mismatch = 1, outputDir = outputDir, overwrite = TRUE)

Best regards,

Julie

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Thanks Julie. The script works using python 2.7. However, changing max.mismatch = 3, gives me an error:

To retain the current behavior and silence the warning, pass 'sort=True'.

featNX = pandas.concat([featNX, NX_onehot], axis=1) Calculates on-target scores for sgRNAs with NGG PAM only. Calculates on-target scores for sgRNAs with NGG PAM only. Calculates on-target scores for sgRNAs with NGG PAM only. Calculates on-target scores for sgRNAs with NGG PAM only. Error in data.frame(..., check.names = FALSE) :
arguments imply differing number of rows: 16, 9

Also, as you confirmed that the lower value of gRNAefficacy indicates lower on-target rate. I was wondering if the range is 0-1? thanks Sharvari

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Sharvari,

I am glad that you got it to work using python 2.7.

Could you please try to run the following script (rule set 1) with max.mismatch = 3 to see if the error goes away? results <- offTargetAnalysis(inputFilePath = gRNAFilePath, findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile,

                     scoring.method = "CFDscore",

                     findPairedgRNAOnly = FALSE, findgRNAs = FALSE,

                     BSgenomeName = Hsapiens, chromToSearch = 'all',

                     txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,

                     orgAnn = org.Hs.egSYMBOL,

                     max.mismatch = 3, outputDir = outputDir, overwrite = TRUE)

Yes, the value of gRNAefficacy is between 0 (lowest efficacy) and 1 (highest efficacy).

Best regards,

Julie

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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Hi Sharvari, Just to let you know that you can set max.mismatch = 0 to speed up the analysis if you are only interested in obtaining the gRNA efficacy. The higher number you set max.mismatch, the slower the analysis takes. Best regards, Julie
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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Hi Sharvari,

Please download Version: 1.25.4 for strand-specific annotation at http://bioconductor.org/packages/devel/bioc/html/CRISPRseek.html. It might take a couple of days for it to propagate. Alternatively, you could download the most recent version using git git clone git@git.bioconductor.org:packages/CRISPRseek

it's correct to interpret the gRNAefficacy value of 0.5 as KO experiment being effective 50%. In your situation, I cannot see why there is a higher read count for KO compared to WT. I suggest you work with local bioinformatician/statistician to see if there is a better way to compare the gene expression level.

Best, Julie

library(CRISPRseek)

library("BSgenome.Hsapiens.UCSC.hg38")

library("org.Hs.eg.db")

library("TxDb.Hsapiens.UCSC.hg38.knownGene")

results <- offTargetAnalysis(inputFilePath = "test.fa",

                     findgRNAsWithREcutOnly = FALSE, 

                     scoring.method = "CFDscore",



                     rule.set = "Root_RuleSet2_2016",



                     findPairedgRNAOnly = FALSE, findgRNAs = FALSE,



                     BSgenomeName = Hsapiens, chromToSearch = 'chr11',



                     txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,



                     orgAnn = org.Hs.egSYMBOL,



                     max.mismatch = 0, outputDir = getwd(), overwrite = TRUE)
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Thanks Julie. I'll download the latest version next week. Btw, is it possible to run the script using rule set 2 and max.mismatches > 3?

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Hi Sharvari, Please download the Version: 1.25.5 of CRISPRseek package to run your script using rule set 2 and max.mismatches > 3. Please let me know if it does not work for you. Best, Julie

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Hi Julie, Using "BiocManager::install("CRISPRseek")" installs version 1.25.3. Can you please let me know how to download the latest version? Thanks

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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Sharvari,

You can either wait for a couple of days for version 1.25.5 to propagate to http://bioconductor.org/packages/devel/bioc/html/CRISPRseek.html, or get the source code using git clone. git clone https://git.bioconductor.org/packages/CRISPRseek Best, Julie
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Hi Julie, Using the latest version 1.25.5, and running the script with rule set 2 and max.mismatch = 3, results in an non-zero gRNAefficacy value for the first hit (n.mismatch = 0), however for all the other hits the extendedSequence and gRNAefficacy values are all NAs. Can you please let me know how to generate non NA values for off-target analysis? Thanks.

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Sharvari, This is intended since gRNA efficacy is only applicable to the on-target. Best regards, Julie

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Thanks Julie. For a couple of sgRNA sequences, 'gene' and 'inExon' columns are empty.I can PM you the gRNA sequence being tested.

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Julie Zhu ★ 4.3k
@julie-zhu-3596
Last seen 6 months ago
United States

Sharvari, I have added a new parameter ignore.strand in the offtargetAnalysis function (default to TRUE). To obtain strand-specific gene annotation, set ignore.strand = FALSE. You can download Version: 1.25.7 using the following command. git clone https://git.bioconductor.org/packages/CRISPRseek Thanks! Best, Julie
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