Prime Editing Guide RNA Design Automation Using PINE-CONE

Kylie Standage-Beier, Stefan J. Tekel, David A. Brafman, Xiao Wang

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.

Original languageEnglish (US)
Pages (from-to)422-427
Number of pages6
JournalACS Synthetic Biology
Issue number2
StatePublished - Feb 19 2021


  • automation
  • genome engineering
  • prime editing

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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