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<p>A standardized ‘parts list’ of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a ‘ten key’ array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.</p>

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A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年11月11日

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  • Troy A. McDiarmid,
  • Megan L. Taylor,
  • Wei Chen,
  • Florence M. Chardon,
  • Junhong Choi,
  • Hanna Liao,
  • Xiaoyi Li,
  • Haedong Kim,
  • Jean-Benoît Lalanne,
  • Tony Li,
  • Jenny F. Nathans,
  • Beth K. Martin,
  • Jordan Knuth,
  • Alessandro L. V. Coradini,
  • Jesse M. Gray,
  • Sudarshan Pinglay,
  • Jay Shendure

摘要

A standardized ‘parts list’ of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a ‘ten key’ array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.