Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

Abstract

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

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

“…To avert this obstacle, strategies have been employed such as using the Cas9 from Staphylococcus aureus (SaCas9 3.2 kb) instead of the commonly used cas Streptococcus pyogenes (SpCas9, 4.2 kb) or using separated AAV vectors for sgRNAs and cas9 . In addition to viral delivery systems, numerous nonviral delivery systems have been developed to facilitate CRISPR/Cas9 delivery including inorganic nanoparticles, lipid nanoparticles, polymer nanoparticles, electroporation, hydrodynamic injection, and microinjection …”

“…146,147 In addition to viral delivery systems, numerous nonviral delivery systems have been developed to facilitate CRISPR/Cas9 delivery including inorganic nanoparticles, lipid nanoparticles, polymer nanoparticles, electroporation, hydrodynamic injection, and microinjection. [148][149][150][151] The major studies highlighted the inhibitory role of CRISPR/Cas9 system on virus replication, although it may induce viral escape. 68,112,113 Mechanistically, CRISPR/Cas9 cleavage generates indel mutations following error-prone NHEJ that may lead to the formation of escape mutants.…”

Delivery of CRISPR/Cas9 for therapeutic genome editing

“…Additionally, they have a reduced risk of immune responses and are free of any risk of insertion into the host genome. At present, a few types of delivery vectors have been exploited for the delivery of genome‐editing agents, such as liposomes, polymers, cell‐penetrating peptides (CPPs), DNA nanostructures, etc …”

“…At present, a few types of delivery vectors have been exploited for the delivery of genome-editing agents, such as liposomes, polymers, cell-penetrating peptides (CPPs), DNA nanostructures, etc. 67,68 In this review, we elaborate first on the modes of CRISPR/Cas9 delivery and the barriers to this process. Secondly, the application of CRISPR/Cas9 in different areas, such as imaging, treatment of diseases and constructions of animal models, is introduced.…”

Geriatrics in the 21st century

“…Clustered Regularly Interspaced Short Palendromic Repeats (CRISPR/Cas9) genomic editing will be mainly used to eradicate congenital diseases at the embryonic stage before birth, but using an adenovirus vector, techniques will emerge to treat sarcopenia, frailty and dementia in older persons (5). This is already being done for the treatment of some cancers (6). An example that should develop over the next decade is using CRISPR/Cas9 to silence the myostatin gene (7).…”