DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons

Biochem Biophys Res Commun. 2020 Apr 9;524(3):621-628. doi: 10.1016/j.bbrc.2020.01.132. Epub 2020 Feb 4.

Abstract

Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the β-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the β-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different β-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR).

Keywords: CRISPR/Cas9; Homology directed repair; In utero electroporation; RAD51; camk2a; β-Actin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Animals
  • Base Sequence
  • Brain / cytology*
  • CRISPR-Cas Systems / genetics*
  • DNA End-Joining Repair*
  • Gene Knock-In Techniques*
  • Green Fluorescent Proteins / metabolism
  • Mice, Inbred ICR
  • Neurons / metabolism*
  • Pyramidal Cells / metabolism
  • RNA, Guide / metabolism
  • Rad51 Recombinase

Substances

  • Actins
  • RNA, Guide
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Rad51 Recombinase