CRISPR Editing Technology in Biological and Biomedical Investigation

Abstract

The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to confer provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future.

Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome

“…However, as viruses have a unique genome with limited similarity to human DNA, usually, off target effects are not considered critical complications. Recently, several modifications have been introduced to enhance the targeting specificity or minimize off‐target cleavage such as using Cas9 nickase instead of Cas9 nuclease, Cas9 protein with altered PAM specific, higher fidelity Cas9 variants, and confining the duration of Cas9 activity …”

“…Recently, several modifications have been introduced to enhance the targeting specificity or minimize off-target cleavage such as using Cas9 nickase instead of Cas9 nuclease, Cas9 protein with altered PAM specific, higher fidelity Cas9 variants, and confining the duration of Cas9 activity. [133][134][135][136][137] Another key obstacle in developing CRISPR/Cas9-based therapeutics is efficient in vivo delivery. A delivery system requires efficient, high tropism for specific cells, with minimal side effects or toxicity.…”

Gene therapy approaches to functional cure and protection of hematopoietic potential in HIV infection

“…However, this study also detected the emergence of escape variant viruses mediated by the host cells' error-prone non-homologous end joining (NHEJ) DNA repair following the CRISPR/Cas9 targeting [87]. The mutagenesis problem with CRISPR/Cas9 has also been observed in treatments of other diseases [88] but can be a serious problem when targeting the HIV DNAs because the strategy might require sustained expression of Cas9 and gRNA in the potential HIV target cells, meaning a sustained risk of mutagenesis [89]. Therefore, an improved method for disrupting HIV DNA while prohibiting the emergence of replication-competent escape variants caused by the NHEJ repair system might be necessary.…”

Innovative Therapeutic Strategies for Cystic Fibrosis: Moving Forward to CRISPR Technique

“…Thanks to this technique it has become possible to intervene on the DNA at a level of precision before impossible, acting with real molecular scissors to cut the helix of the DNA at the desired point and replace a stroke ( White et al, 2017 ). The repair of a defective gene at its original locus has two major advantages.…”