Gigabase-scale deletion scanning of the human genome

作者：Jonas Koeppel, Aidan Keith, Samantha Sgrizzi, PeiXi Chen, Riza Maza Daza, Faaiz Quaisar, Eleftheria Anastasia, Zihao Song, Jay Shendure, Sudarshan Pinglay · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2026 · DOI：10.64898/2026.05.29.728882 · 研究领域：Single-cell and spatial transcriptomics、CRISPR and Genetic Engineering、Cell Image Analysis Techniques

ABSTRACT What fraction of the human genome is essential for cellular viability? To date, essentiality in human cells has been mapped almost exclusively at the level of individual open reading frames (ORFs). Whether noncoding regions and broader architectural features of the genome are required for human cells to remain viable, and where the boundaries of any such regions lie, remains largely unexplored. Here we introduce Shred-seq, which couples Type I-C CRISPR-Cas3-mediated deletions with phage polymerase-based genotyping to enable large-scale deletion scans of the human genome. Shred-seq leverages thousands of genomically integrated, mapped target sites as launchpads for Cas3 to initiate unidirectional deletions ranging in size from hundreds of base pairs (bp) to hundreds of kb. Breakpoints are directly captured at high resolution by in vitro or in situ transcription from flanking phage polymerase promoters, enabling bulk or single-cell phenotyping, respectively. In this proof-of-concept, we generate and genotype 36,257 independent deletions originating from 9,604 Cas3 launchpads, individually spanning 100 bp to 500 kb, collectively covering 461 Mb (14% of the human genome), and totaling to 2.55 Gb of deleted sequence (∼10-fold coverage of these regions). Surviving deletions are depleted not only for essential protein-coding genes, but also for active, conserved and mutation-constrained non-coding sequences, directly quantifying purifying selection across both coding and no...