Third-party CRISPR Guide Assignment Toolkit

Peer-Reviewed Option for performing CRISPR gRNA thresholding

Crispat is a peer-reviewed CRISPR guide calling package that conveniently bundles many popular gRNA thresholding approaches, enabling users to compare the performance of multiple approaches side-by-side.

Here, we implement one of these approaches, the Gaussian-Gaussian Mixture Model, which we've found performs best with the Illumina's Single-Cell CRISPR Prep kit.

Instructions for how to download and install our optimized fork of crispat can be found here.

In brief, the model does the following:

• Read in DRAGEN filtered matrix files from an DRAGEN output directory.

• Creates and reads in a .h5ad file (via scanpy).

• Performs 2-component Gaussian-Gaussian mixture modeling on the gRNA counts.

• Outputs gRNA assignments for each cell barcode along with plots showing the fit of the model to the raw counts.

A step-by-step tutorial and a small test dataset can be found in the repo.

You should expect the plots of the models fit to nonzero count data to look similar to this:

The output file assignments.csv provides a full list of all cell barcodes containing a guide that exceeded the threshold determined by the gaussian mixture model. The outputs should look as follows:

cell: the cell barcode sequence.

gRNA : the gRNA ID that was assigned to the cell.

read_counts: the number of read counts observed for each cell-gRNA assignment.