CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly

Methods Mol Biol. 2020:2115:365-383. doi: 10.1007/978-1-0716-0290-4_20.

Abstract

CRISPR Cas9 genome editing allows researchers to modify genes in a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within 6 years of its initial application, CRISPR-Cas9 genome editing has been widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects, need careful consideration. Obtaining custom donor vectors can also be expensive and time-consuming. This chapter details strategies to overcome barriers to CRISPR-Cas9 genome editing as well as recent developments in employing this technique.

Keywords: CRISPR; FOXO3; Gene editing; Gibson assembly.

MeSH terms

  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Forkhead Box Protein O3 / genetics
  • Gene Editing / methods*
  • Genetic Vectors / genetics
  • Humans
  • Mutation
  • RNA, Guide, CRISPR-Cas Systems / genetics

Substances

  • Forkhead Box Protein O3
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9