Progress of delivery methods for CRISPR-Cas9

Abstract

Introduction: Gene therapy is becoming increasingly common in clinical practice, giving hope for the correction of a wide range of human diseases and defects. The CRISPR/Cas9 system, consisting of the Cas9 nuclease and single-guide RNA (sgRNA), has revolutionized the field of gene editing. However, how to efficiently deliver the CRISPR-Cas9 to the target organ or cell remains a significant challenge. In recent years, with rapid advances in nanoscience, materials science, and medicine, researchers have developed a variety of technologies that can deliver CRISPR-Cas9 in different forms for in vitro and in vivo gene editing. Here, we review the development of the CRISPR-Cas9 and describe the delivery forms and the vectors that have emerged in CRISPR-Cas9 delivery, summarizing the key barriers and the promising strategies that vectors currently face in delivering the CRISPR-Cas9. Areas covered:With the rapid development of CRISPR-Cas9, delivery methods are becoming increasingly important in the in vivo delivery of CRISPR-Cas9.Expert opinion: CRISPR-Cas9 is becoming increasingly common in clinical trials.However, the complex nuclease and protease environment is a tremendous challenge for in vivo clinical applications. Therefore, the development of delivery methods is highly likely to take the application of CRISPR-Cas9 technology to another level.

How to detect CRISPR with CRISPR – employing SHERLOCK for doping control purposes

“…in vivo or ex vivo approaches, systemic or local administration, viral or non-viral delivery and CRISPR/ Cas format [RNP, mRNA or DNA] need to be made. 38 This overwhelming multitude of parameters that need to be considered, also in terms of anti-doping analysis, complicate the detection if illicitly used CRISPR/Cas. Therefore, this study demonstrates just one of many complementary analysis methods that need to be developed in future work to ensure the overall detection of gene doping with CRISPR/Cas.…”

Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases

“…This new approach is more attractive concerning HDR since NHEJ is always active in the cell cycles but, at the same time, it brings many off-target modifications and the gene knock-in efficiency is less than 5%. Following recent breakthroughs such as Microhomology-mediated end joining, or the introduction of point mutations into either of the Cas9 nuclease domains which allows single-stranded nicks instead of double-stranded breaks, CRISPR-Cas is becoming an increasingly precise and smart genome editing tool [ 28 ].…”

“…Indeed, to perform its editing function, the CRISPR-Cas9 system needs to overcome different biological barriers. Moreover, CRISPR-Cas9 requires the codelivery of Cas9 protein and the sgRNA, which can be achieved in three different ways [ 28 , 38 ]: Cas9 protein and sgRNA : The most straightforward approach of CRISPR-Cas9 delivery consists of the RNP complex derived from the in vitro incubation of Cas9 protein and sgRNA. This CRISPR-Cas9 form is ready for genome editing and, the short half-life of the Cas9 protein allows the reduction of off-target modifications.…”

“…Cellular uptake and endosomal escape of these DNA-based nanostructures were enhanced by coating the carriers with the cationic polymer PEI resulting in a genome editing efficiency of approximately 28%. Despite general improvements in CRISPR-Cas9 delivery over the years, further investigations regarding cytotoxicity and immunogenicity are required [ 28 ].…”

CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology

“…Hydrodynamic delivery is an in vivo delivery approach that includes quickly injecting a large volume (nearly 8–10% body weight) of fluid carrying the gene editing cargo into an animal's circulation, mostly through the tail vein in mice. Since, blood is incompressible, the huge bolus of liquid causes a rise in hydrodynamic pressure, which momentarily increases permeability into endothelium and parenchymal cells, allowing the cargo (naked DNA, plasmid and proteins) to penetrate a cellular membrane and enter into the cells [ 21 ]. This form of cargo delivery is prevalent in the liver, but also includes cells from the kidneys, lungs, muscles, and heart.…”