Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

Abstract

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

Non-viral delivery of the CRISPR/Cas system: DNAversusRNAversusRNP

“…Synthetic sgRNA which lacks a 5′-triphosphate group is less immunogenic and does not induce detectable immune response in many cell types. [66][67][68] Moreover, the chemical oligonucleotide assembly enables site-specific incorporation of modified nucleotides during solid-phase synthesis. Various chemically modified nucleotides, including 2′-methyl (M), 2′-O-methyl-PS (MS), 2′-O-methyl-3′-thiophosphonoacetate (MSP), phosphorothioates, 2′-fluoro (F), 2′-O-methyl-3′-phosphonoacetate (MP), locked nucleic acids (LNA) and bridged nucleic acids (BNA), are developed to enhance the enzymatic stability, editing efficiency, specificity as well as to reduce the immunogenicity and off-target effects of sgRNA.…”

Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy

“…Cas9 is an RNA-guided endonuclease that recognizes and cleaves target DNA that have template strand pairing to the guide RNA, which is composed of Crispr RNA (crRNA) and tracrRNA [ 13 ]. crRNA, which has a [ 17 – 20 ] nucleotide sequence that is complementary to the target DNA, and tracrRNA, which acts as a Cas nuclease binding scaffold [ 14 ].…”

Viral Vectors for the in Vivo Delivery of CRISPR Components: Advances and Challenges

“…The crRNA is an 18–20 base pair RNA complementary to the target DNA, whereas tracrRNA is a lengthy stretch of loops that serves as a Cas nuclease binding scaffold ( Marx, 2020 ). It can be synthesized by combining crRNA and tracrRNA to generate a single guide RNA (sgRNA) to target the gene sequence in the gene-editing tool ( Allen et al, 2021 ). Even though CRISPR/Cas9 has numerous roles ranging from basic molecular research to clinical applications, one of the current challenges of this technology is the lack of safe and effective delivery methods ( Asmamaw and Zawdie, 2021 ).…”