Optimization of genome editing through CRISPR-Cas9 engineering

Abstract

CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

Keywords: CRISPR-Cas9; efficiency; genome editing; specificity.

Figure 1.

Schematic diagram of the functional mechanism and structural conservation of CRISPR-Cas9. (A) gRNA interrogation of target protospacer DNA created according to PDB ID:5F9R. The heteroduplex of gRNA and the target strand of the protospacer DNA is bound in the primary binding channel formed between the REC and NUC lobes; the non-target strand of the protospacer DNA is bound in the minor binding channel formed within the NUC lobe; the 2 HNH and RuvC endonuclease domains, the PI domain and PAM site are also indicated. (B) SaCas9 and gRNA complex highlighting the PAM and PAM interacting amino acids of the PI domain, created according to PDB ID: 5CZZ. Seven colors represent different amino acids. (C) SpCas9 and gRNA complex highlighting the PAM and PAM interacting amino acids of the PI domain created according to PDB ID: 4UN3. Five colors represent 5 different amino acids. (D) Interacting forces among the gRNA (Z), target DNA strand (Y), non-target DNA strand (X) and amino acids in the minor binding channel (Wn). Fyz represents hydrogen bonds of the pairing nucleotides between gRNA and target DNA strand of the protospacer DNA; Fxy represents the hydrogen bonds of the pairing nucleotides between the target and non-target strands of the protospacer DNA; Fwx represents the pulling force generated from the minor binding channel and asserting on the non-target strand of the protospacer DNA. (E) Phylogenetic tree of CRISPR-Cas9s (ConSurf and Phylogeny.fr) from 58 bacterial species showing distant evolutionary relations between SpCas9 and SaCas9. The 3 letters of the genus names were adopted only for the purpose of distinctions. (F) Weblogo display of the amino acid sequence homologies represents discrete homologous and non-homologous regions among the 58 CRISPR-Cas9s described in E. The color represents amino acid property and the size corresponds to relative degree of conservation.