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

Genome Biol. 2021 Mar 16;22(1):83. doi: 10.1186/s13059-021-02304-3.


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.

Keywords: CRISPR; Gene expression; Genome editing; Mouse; Prime editing; Transcription.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • CRISPR-Cas Systems*
  • Fluorescent Antibody Technique / methods
  • Gene Editing* / methods
  • Gene Expression Regulation*
  • Mice
  • Mice, Transgenic
  • Nerve Tissue Proteins / genetics
  • Organ Specificity / genetics
  • Point Mutation*
  • Promoter Regions, Genetic
  • Protein Binding
  • Recombinational DNA Repair
  • Tetraspanins / genetics


  • Nerve Tissue Proteins
  • Tetraspanins
  • Tspan2 protein, mouse