Casper: Algorithms and Integrated Software Platform for Precise CRISPR Genome Editing of Single and Consortia of Organisms

Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation in synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions. To accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed algorithms and associated software, named CASPER (CRISPR Associated Software for Pathway Engineering and Research), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, p<0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA (gRNA) requirement) and multispecies population analysis (i.e. gRNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. Utilizing multiple algorithms for on- and off-target analysis of the guided RNA/Cas complex, CASPER achieves higher accuracy at predicting experimental results. Further, CASPER enables end-users to design guided RNAs to target multiple chromosome loci at once and across a consortium of organisms. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.