CRISPR-HAWK: Haplotype- and Variant-Aware Guide Design Toolkit for CRISPR-Cas

Motivation Current CRISPR guide RNA design tools rely on reference genomes, overlooking how genetic variation impacts editing outcomes. As genome editing advances toward clinical applications, incorporating population diversity becomes essential for ensuring therapeutic efficacy across diverse populations. Results We present CRISPR-HAWK, a framework integrating individual- and population-scale variants and haplotypes into gRNA design. Analyzing therapeutic targets across 79,648 genomes reveals that genetic variants substantially alter guide performance. For the clinically approved sickle cell disease therapeutic guide targeting BCL11A, we identify haplotypes that completely abolish predicted cutting activity. Across seven therapeutic loci, 82.5% of guides contain variants modifying on-target activity. Variants also create novel protospacer adjacent motif sites generating individual-specific guides invisible to reference-based design. These findings demonstrate that variant-aware selection is critical for equitable genome editing.