Hello everyone,

i want to adjust some features within the CRISPRseek package for Cpf1. Can somebody tell me more about the Argument gRNA.backbone and the subPAM.activity ? I don't know how the arguments work and need a further description as in the paper Package "CRISPRseek" from May 26. Maybe somebody knows where i can get those further descriptions or can help me with the arguments. I have already adjusted the other parameters and was able to compute gRNA sequences for Cpf1 and even test them successful in vitro. Maybe i can get better results with the adjusted arguments.

Additionally I don't know which rule set i could use for calculating the most accurate gRNAefficiacy. Is CRISPRscan an option?

I would appreciate your help and hope you can help me with my request.

Kind regards,

Nils

Hi Nils,

Sorry for having missed this post!

gRNA.backbone refers to the constant region sequence with default to the sequence in Sp gRNA backbone. It is no longer used due to the deprecation of the dependent package GeneRFold for calculating minimum free energy of the secondary structure.

subPAM.activity is only applicable only when scoring.method is set to CFDscore with spCas9 system. It represents the cleavage rate for each alternative sub PAM sequence relative to preferred PAM sequence

For detailed description of other parameters in OffTargetAnalysis function, please type help(offTargetAnalysis) in a R session.

Best regards, Julie

Hi Nils,

Sorry for having missed this post!

gRNA.backbone refers to the constant region sequence with default to the sequence in Sp gRNA backbone. It is no longer used due to the deprecation of the dependent package GeneRFold for calculating minimum free energy of the secondary structure.

subPAM.activity is only applicable only when scoring.method is set to CFDscore with spCas9 system. It represents the cleavage rate for each alternative sub PAM sequence relative to preferred PAM sequence

Yes, CRISPRscan algorithm has been implemented in CRISPRseek. Please see the section 2.7 at https://www.bioconductor.org/packages/release/bioc/vignettes/CRISPRseek/inst/doc/CRISPRseek.pdf. However, I believe this algorithm is applicable to spCas9 with NGG PAM.

For detailed description of other parameters in OffTargetAnalysis function, please type help(offTargetAnalysis) in a R session.

Best regards, Julie