High-Coverage Whole-Genome Sequencing Uncovers Complex CRISPR-Induced Genomic Rearrangements in Human Hematopoietic Stem Cells

CRISPR-Cas genome editing introduces genomic alterations at on- and off-target sites, requiring thorough characterization for safe clinical use, including small insertions and deletions (indels) and structural variants (SVs), such as large deletions, inversions, and translocations. These events can compromise genome integrity and potentially drive tumorigenesis. Current methods rely on targeted deep sequencing, effective for small indels but limited in identifying large or unexpected rearrangements.

Whole-genome sequencing (WGS) offers comprehensive, unbiased analysis but is constrained by cost and depth. To address this, we employed Ultima Genomics` platform, achieving 1000× WGS at reduced cost, to profile CRISPR-edited human hematopoietic cells targeting HBB, HBG, and BCL11A (used in CASGEVY, the first FDA-approved CRISPR therapy). Our genome-wide pipeline identified diverse CRISPR-induced alterations.

High-coverage WGS revealed a complex spectrum of events at on-target loci, including small indels and deletions spanning from 200 bp to several kilobases. Notably, 1–4% of sequencing reads showed chromosomal translocations involving targeted loci, mostly at unexpected sites, highlighting unforeseen genomic impacts. We also observed complex intra-chromosomal SVs; notably, the HBG gRNA induced inversions (~5 kb) in over 5% of edited alleles—events missed by targeted sequencing. Additionally, structural rearrangements were detected at known off-target sites like OT1 for HBB.

Comparisons confirmed targeted sequencing detects short deletions (<200 bp), but only WGS captures larger deletions and complex, unanticipated translocations. These findings emphasize the critical importance of unbiased, high-resolution genomic tools, underscoring the potential of streamlined, WGS-based strategies in comprehensive safety assessments for CRISPR-based therapies.