Combining CRISPR with Flow‐FISH to study CRISPR‐mediated genome perturbation

Abstract

Since the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system as a genome editing tool, the ease of studying gene function and the impact thereof on cellular function has increased incrementally. Not surprisingly, the original describers of the CRISPR/Cas system received the 2020 Nobel Prize in Chemistry. Compared to conventional genome editing tools such as Transcription Activator-Like Effector Nucleases (TALENs) or Zinc Finger Nucleases (ZFNs), CRISPR is a more versatile platform that can be easily adjusted to target new genes of interest.The mechanism behind genome editing by the CRISPR/Cas9 system has been recently thoroughly reviewed elsewhere [1]. Briefly, CRISPR-mediated genome editing is dependent on at least two components: (1) a Cas protein that possesses endonuclease activity and(2) a variable 20 base pair nucleic-acid based targeting crisprRNA (crRNA) that defines the target of interest. Depending on the type of Cas protein employed, also a trans-activating RNA (tracrRNA) is required in order to activate nuclease activity. Together, the gRNA and tracrRNA are often referred to as the single guide RNA, or sgRNA.