The CRISPR/Cas9 system has been developed as a powerful tool for targeted gene editing. As a result of technical enhancements in recent years, this technology has become the method of choice for efficiently modifying targeted HIV-1 genome efficiently as part of HIV therapy. CRISPR can be modified to target specific sequences that Cas9 then cuts. In this article, we outline the development of the CRISPR/Cas9 system. We also show how this technology can be used for the prevention and treatment of HIV-1 infection. Optimistically, this technology promises to make a significant impact on the fight against HIV-1 in the future.

External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5 ′ ′ ′ ′ ′ termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase Pmediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%-74% and a decrease of viral growth by about 10,000-and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.

Pigs provide valuable meat sources, disease models, and research materials for humans. However, traditional methods no longer meet the developing needs of pig production. More recently, advanced biotechnologies such as SCNT and genome editing are enabling researchers to manipulate genomic DNA molecules. Such methods have greatly promoted the advancement of pig research. Three gene editing platforms including ZFNs, TALENs, and CRISPR/Cas are becoming increasingly prevalent in life science research, with CRISPR/Cas9 now being the most widely used. CRISPR/Cas9, a part of the defense mechanism against viral infection, was discovered in prokaryotes and has now developed as a powerful and effective genome editing tool that can introduce and enhance modifications to the eukaryotic genomes in a range of animals including insects, amphibians, fish, and mammals in a predictable manner. Given its excellent characteristics that are superior to other tailored endonucleases systems, CRISPR/Cas9 is suitable for conducting pig-related studies. In this review, we briefly discuss the historical perspectives of CRISPR/Cas9 technology and highlight the applications and developments for using CRISPR/Cas9-based methods in pig research. We will also review the choices for delivering genome editing elements and the merits and drawbacks of utilizing the CRISPR/Cas9 technology for pig research, as well as the future prospects.