Scholarlink

<p>CRISPR-associated proteins (Cas) are central to gene editing, forming nuclease complexes with guide RNA to enable precise genome modification. Among numerous Cas variants, Cas9 and Cas12a are the most extensively studied. While much is known about the genomic substrates for these enzymes, less is known about the determinants of the DNA cleavage activity. Wild-type Cas12a exhibits higher intrinsic specificity than Cas9, minimizing off-target activity, but lower overall potency. Recent protein engineering has sought to improve both parameters. Here, we shed light on the structural and mechanistic basis by which an engineered AsCas12a variant achieves high potency while retaining its hallmark specificity. We show that reduced protein–DNA interactions facilitate more rapid R-loop formation, thereby enhancing cleavage activity. These results provide mechanistic insight into Cas12a function and highlight strategies for designing genome-editing nucleases with optimal balance between efficiency and specificity.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Mechanistic basis for improved activity of Engineered AsCas12a

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年3月10日

查看全文
  • Linnea Jansson-Fritzberg,
  • Bryant Chica,
  • Chrysa Latrick,
  • Andrea Olland,
  • Alexey Dementiev,
  • Andre White,
  • Steffen Kutter,
  • Jean-Noel Lemercier,
  • Steven Wolk

摘要

CRISPR-associated proteins (Cas) are central to gene editing, forming nuclease complexes with guide RNA to enable precise genome modification. Among numerous Cas variants, Cas9 and Cas12a are the most extensively studied. While much is known about the genomic substrates for these enzymes, less is known about the determinants of the DNA cleavage activity. Wild-type Cas12a exhibits higher intrinsic specificity than Cas9, minimizing off-target activity, but lower overall potency. Recent protein engineering has sought to improve both parameters. Here, we shed light on the structural and mechanistic basis by which an engineered AsCas12a variant achieves high potency while retaining its hallmark specificity. We show that reduced protein–DNA interactions facilitate more rapid R-loop formation, thereby enhancing cleavage activity. These results provide mechanistic insight into Cas12a function and highlight strategies for designing genome-editing nucleases with optimal balance between efficiency and specificity.