Improving CRISPR Genome Editing by Engineering Guide RNAs

• Published in: Trends in biotechnology (Regular ed.) 2019-08, Vol.37 (8), p.870-881
• Main Authors: Moon, Su Bin, Kim, Do Yon, Ko, Jeong-Heon, Kim, Jin-Soo, Kim, Yong-Sam
• Format: Article
• Language: English
• Subjects: Binding sites; Chemical synthesis; Clinical trials; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; Deoxyribonucleic acid; DNA; Efficiency; Endoribonucleases - metabolism; Engineering; FDA approval; Flexibility; Gene Editing - methods; Gene Editing - trends; Gene expression; Gene targeting; Gene therapy; Genetic Engineering - methods; Genetic Engineering - trends; Genetic modification; Genome editing; Genomes; guide RNA; guide RNA engineering; Multiplexing; Nucleotide sequence; Organic chemistry; Proteins; Ribonucleic acid; RNA; RNA editing; RNA polymerase; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Spacer; Substrate Specificity; Toxicity; Transcription
• ISSN: 0167-7799; 1879-3096
• DOI: 10.1016/j.tibtech.2019.01.009

CRISPR technology is a two-component gene editing system in which the effector protein induces genetic alterations with the aid of a gene targeting guide RNA. Guide RNA can be produced through chemical synthesis, in vitro transcription, or intracellular transcription. Guide RNAs can be engineered to have chemical modifications, alterations in the spacer length, sequence modifications, fusion of RNA or DNA components, and incorporation of deoxynucleotides. Engineered guide RNA can improve genome editing efficiency and target specificity, regulation of biological toxicity, sensitive and specific molecular imaging, multiplexing, and editing flexibility. Therefore, engineered guide RNA will enable more specific, efficient, and safe gene editing, ultimately improving the clinical benefits of gene therapy. Independently from engineering effector proteins, such as Cas9 and Cpf1, the engineering of guide RNAs themselves has provided useful and versatile options to improve the CRISPR technology as a genome editing toolset. Guide RNAs can be prepared by chemical synthesis, in vitro transcription, or intracellular transcription systems. Guide RNAs can be engineered in several ways, including chemical modifications, alterations in the spacer length, sequence modifications in the spacer or scaffold, fusion with additional DNA or RNA components, and partial replacement with DNA. The engineered guide RNAs contribute to improved genome editing efficiency and target specificity, regulation of biological toxicity, sensitive and specific molecular imaging, multiplexing, and genome editing flexibility.