Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

Pan Gao, Qing Lyu, Amr R. Ghanam, Cicera R. Lazzarotto, Gregory A. Newby, Wei Zhang, Mihyun Choi, Orazio J. Slivano, Kevin Holden, John A. Walker, Anastasia P. Kadina, Rob J. Munroe, Christian M. Abratte, John C. Schimenti, David R. Liu, Shengdar Q. Tsai, Xiaochun Long, Joseph M. Miano

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


Background: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Original languageEnglish (US)
Article number83
Pages (from-to)83
JournalGenome Biology
Issue number1
StatePublished - Dec 2021


  • Gene expression
  • Genome editing
  • Mouse
  • Prime editing
  • Transcription

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology


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