In the last decade

...there have been a variety of papers published that suggested nucleosome presence lessens Cas9's ability to make a double stranded break at a specific target site, especially in yeast. In light of this information, few if any gRNA design tools have been modified to account for this. This gRNA design tool helper for the eukaryotic model organism

S. cerevisiae

is meant to help researchers evaluate potential gRNA targets more completely and contribute to an increased efficiency in CRISPR-Cas9 experiments. Please note that at this time our tool is still being validated. As such there may be some bugs we've yet to work out. If you run into some kind of issue while using this tool that you believe is bug-related, please contact us.

The Nucleosome Maps & Filtering Methods

The two nucleosome maps used in creating this tool are taken from this publication by Kristin Brogaard, Liqun Xi, Ji-Ping Wang and Jonathan Widom. They are:

(1) a unique map in which nucleosome domains are not allowed to overlap by more than a specified number of base pairs

(2) redundant map in which nucleosomes may arbitrarily overlap each other.

In all of our filtering methods, the returned gRNAs are treated categorically, designated full or partially occupied, or unoccupied. In the dynamic sort fully occupied gRNAs are returned in order of their original ranks while partially occupied gRNAs are re-ordered based on the NCP Score:Noise ratio and original rank. In the strict sort, all nucleosomes from both maps are treated as having absolute inaccessibility. As such any gRNA overlapping with any nucleosome via this method is designated fully occupied, and rank is maintained among occupied gRNAs based on original ordering of gRNAs. In the lenient sort method, only the information from the unique nucleosome map is used and all gRNAs overlapping with nucleosomes are considered only partially occupied. gRNAs filtered via this method are re-ranked basked on the NCP Score:Noise Ratio and original rank. For more information regarding our filtering methods, please see documentation in our github repo here.

Tool Validation

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