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<p>Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the <i>cis</i>-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their <i>cis</i>- and <i>trans</i>-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.</p>

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Mapping cis- and trans-regulatory target genes of human-specific deletions

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月20日

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  • Tyler Fair,
  • Bryan J. Pavlovic,
  • Dani Swope,
  • Octavio E. Castillo,
  • Nathan K. Schaefer,
  • Alex A. Pollen

摘要

Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.