Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Abstract

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

Engineering and Design of Programmable Genome Editors

“…Most of the increases in the functional efficiency of the current genome editors have occurred by utilization of experimental molecular protein engineering approaches such as directed evolution and selection with random mutagenesis, combinatorial mutagenesis, and phage display; structural knowledge and molecular dynamics simulations have played a significant but relatively minor role. , Molecular methods for overcoming the limitations of the classical genome editors and for enhancement of the precision of genomic and epigenomic editing have been reviewed recently. − Further increases in specificity and efficiency of genome editing may be possible by leveraging the accumulated biophysical knowledge and the recent advances in protein stability, folding, and dynamics. − This review emphasizes the experimental and computational studies that have provided valuable insights regarding the relevant structures of protein–DNA complexes, conformational dynamics involved in the scanning and binding events, DNA unwinding, protein folding, and unfolding. − Computational design and engineering of sequence-specific DNA binding proteins is complicated by the structural plasticity of DNA and the altered conformations of histone-bound DNA in vivo . , Biophysical characterization of the structure and dynamics of the on-target and off-target activities of native and engineered genome editors reveal an intricate interplay of conformational changes involving both the DNA and the protein, which may involve partial protein unfolding and folding and partial unwinding of DNA, which produce a choreographed sequence of events resulting in the scanning of millions of base pairs, selection of target DNA sequence, and enzyme catalyzed site-specific DNA modifications. , Choreography of the “dance” involving the concerted conformational changes in the target DNA and the designed proteins is the key to the rational engineering and design of genome editors with improved efficiency…”

Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts

“…The required concentrations of small molecules for precise gene editing were determined through cytotoxicity experiments and a literature review, which were based on the effect of small molecule concentrations on cell viability and CRISPR-mediated gene editing efficiency [ 19 , 27 ]: L-189 at 5 μM; NU7441 at 1 μM; SCR7 at 10 μM; L755507 at 5 μM; RS-1 at 10 μM; and Brefeldin A at 0.5 μM ( Figure 2 ).…”

Zebrafish CCNF and FUS Mediate Stress-Specific Motor Responses

“…The CRISPR/Cas9 system offers simplicity and is versatility, and relatively low cost, and the TALEN system provides a more customizable approach with potentially higher specificity [ 34 ]. The applications of different genome editing strategies are discussed in greater detail by Denes et al [ 35 ].…”