Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells

Methods Enzymol. 2019;616:241-263. doi: 10.1016/bs.mie.2018.10.028. Epub 2019 Jan 17.

Abstract

CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.

Keywords: CRISPR; Cas; Cas12a; Cas9; Embryonic stem cells; Genetic engineering; Genome editing.

MeSH terms

  • Animals
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems*
  • Electroporation / methods
  • Gene Deletion*
  • Gene Editing / methods*
  • Genetic Engineering / methods
  • Mice
  • Mouse Embryonic Stem Cells / metabolism*
  • RNA, Guide / genetics

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide