A Split CRISPR-Cpf1 Platform for Inducible Gene Activation

Methods Mol Biol. 2023:2577:229-240. doi: 10.1007/978-1-0716-2724-2_16.

Abstract

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.

Keywords: CRISPR–Cpf1 (also known as Cas12a); Gene activation; Genome editing; Genomic PCR; Hydrodynamic tail vein injection; In vivo gene activation; NHEJ; Optogenetics; T7E1; crRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Endonucleases* / genetics
  • Endonucleases* / metabolism
  • Gene Editing* / methods
  • Genome
  • Mammals / metabolism
  • Mice
  • RNA
  • Transcriptional Activation

Substances

  • RNA
  • Endonucleases