title: 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.
authors.affiliation: a Metabolic Diseases Branch , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Zhang
authors.affiliation: a Metabolic Diseases Branch , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Poorni
authors.initials: P
authors.lastname: Adikaram
authors.affiliation: a Metabolic Diseases Branch , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Mritunjay
authors.initials: M
authors.lastname: Pandey
authors.affiliation: a Metabolic Diseases Branch , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda , MD , USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Genis
authors.affiliation: a Metabolic Diseases Branch , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda , MD , USA.
authors.forename: William F
authors.initials: WF
authors.lastname: Simonds
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D016678:Genome; D006801:Humans; D008958:Models, Molecular; D059372:Nucleotide Motifs; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR-Cas9; efficiency; genome editing; specificity