Optimized sgRNA design to maximize activity and minimize off-target effects for genetic screens with CRISPR-Cas9

CRISPR technology has proven to be a powerful means of conducting genetic screens. The ease of programming the Cas9 protein with an sgRNA presents an abundance of potential target sites for each gene, but the on-target activity and off-target effects of individual sgRNAs can vary. Here, we incorporated recently devised sgRNA design rules to create human and mouse genome-wide libraries, performed positive and negative selection screens, and observed that the use of these rules greatly enhances the consistency and depth of screening results. Additionally, we profiled the off-target activity of thousands of sgRNAs and developed a metric to predict off-target sites that outperforms existing metrics. We combine these findings from large-scale, empirical data to improve our computational design rules and create highly-optimized sgRNA libraries that maximize on-target activity and minimize off-target effects in order to enable more effective and efficient genetic screens and genome engineering.