Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

Keywords: CRISPR efficiency; homology directed repair; low molecular weight compounds.

Figure 1

Major mammalian DNA damage repair pathways at Cas9-induced DSBs together with small molecules and one peptide (i53) reported to increase knock-in efficiencies. Shown are the three major repair pathways after a CRISPR/Cas9-induced DNA double-strand break. (a) Depicted is a Cas9/sgRNA complex cleaving DNA. (b) During Non-Homologous End-Joining (NHEJ) Ku70/Ku80 protect free DNA-ends from end resection. DNA-Protein-Kinase catalytical subunit (DNA-PKcs) phosphorylates different DNA repair enzymes. Ends are processed through Artemis, Polymerase Mu (POLM) and Polymerase Lambda (POLL) and ligated by the Ligase IV, X-Ray Repair Cross-Complementing Protein and 4 XRCC4-like Factor (LIG4-XRCC4-XLF) complex. (c,d) Breast Cancer Type 1 (BRCA1) antagonizes p53-Binding Protein 1 (53BP1) and enables end resection mediated by CtBP-interacting protein (CtIP) and the MRN complex Meiotic Recombination 11 (MRE11), RAD50, and Nijmegen Breakage Syndrome 1 (NBS1) necessary for alternative End-Joining (a-EJ) and Homology Directed Repair (HDR). The Kinases Ataxia Telangiectasia Mutated (ATM) and ATM-Rad3- related (ATR) function as damage sensors and activate different repair enzymes. (c) In a-EJ extensive end resection is prevented through Poly [ADP-ribose] Polymerase 1 (PARP1). After annealing of short homologies, X-Ray Repair Cross Complementing 1 (XRCC1) or Flap Endonuclease 1 (FEN1) cleave 5′-flaps and Polymerase Theta (POLQ) performs gap-fillings. Ligase I (LIG1) or Ligase III alpha-XRCC1 (LIGA-XRCC1) ligate DNA ends. (d) HDR requires extensive end resection mediated by Exonuclease 1 (EXO1) or Bloom Helicase and DNA2 Helicase/Nuclease (BLM-DNA2). Replication Protein A (RPA) binding of single-stranded DNA prevents the formation of secondary structures. RPA is replaced by RAD51 with the help of Breast Cancer type 2 (BRCA2) and Partner and Localizer of BRCA2 (BRAC2-PALB2). RAD51 promotes homology donor search and base pairing. (e) Cell cycle dependency of DNA repair pathways: NHEJ is active through all cell cycle phases. Pathways requiring end resection are mainly active in the S-G2 phase.