Evolution of CRISPR towards accurate and efficient mammal genome engineering

Abstract

The evolution of genome editing technology based on CRISPR (clustered regularly interspaced short palindromic repeats) system has led to a paradigm shift in biological research. CRISPR/Cas9-guide RNA complexes enable rapid and efficient genome editing in mammalian cells. This system induces double-stranded DNA breaks (DSBs) at target sites and most DNA breakages induce mutations as small insertions or deletions (indels) by non-homologous end joining (NHEJ) repair pathway. However, for more precise correction as knock-in or replacement of DNA base pairs, using the homology-directed repair (HDR) pathway is essential. Until now, many trials have greatly enhanced knock-in or substitution efficiency by increasing HDR efficiency, or newly developed methods such as Base Editors (BEs). However, accuracy remains unsatisfactory. In this review, we summarize studies to overcome the limitations of HDR using the CRISPR system and discuss future direction. [BMB Reports 2019; 52(8): 475-481].

Fig. 1

Small molecules enhance knock-in efficiency. (A) Small molecules related to the NHEJ or HDR repair pathway. Inhibitors are labeled in red, activators are labeled in blue. NU7026 inhibits DNA-PK, and SCR7, E1B55K, and E4orf6 inhibit DNA ligase IV. MLN4924, NSC15520, RS-1, Trichostatin A, or Resveratrol enhance CtIP, RPA, RAD51, or ATM, respectively. ATM protein also induces activation of RPA, BRCA2, and RAD51. The i53 is an inhibitor of 53BP1. The i53 activates DNA end resection and recruitment, of BRCA1 to DSBs. (B) HDR activity is increased at S/G2 phase. NHEJ activity is labeled in blue, HDR activity is labeled in red. Small molecules are used to arrest the cell cycle at specific phase, to improve HDR efficiency. L755505, Resveratrol, Mimosine, Aphidicolin, Thymidine and Hydroxyurea block cells at the G1 to S phase before DNA replication, and Nocodazole arrests cell cycle at G2/M phase. Lovastatin also inhibits at early G1, and partially at G2/M phase.

Fig. 2

Schematics of base editors (BEs). (A) The cytidine base editor (CBE) consists of cytidine deaminase rAPOBEC1 (blue), uracil glycosylase inhibitor (UGI) and nickase Cas9 (nCas9) or dead Cas9 (dCas9). CBE can induce targeted nucleotide substitutions, such as C to T, or G to A conversion. (B) The adenine base editor (ABE) consists of adenine deaminase TadA (orange,) and nCas9 or dCas9. ABE can induce targeted nucleotide substitutions, such as A to G, or T to C conversion. The active window of CBE and ABE is 4–8 nucleotides, in the distal region of the guide RNA.