• Nanotechnology-based delivery of CRISPR/Cas9 system for genome editing
  • Hossein Rahimi,1 Navid Mousazadeh,2 Motahare Sharifyrad,3 Fina Abdoli,4 Ali Sadeghi,5 Seyed Sadegh Eslami ,6,*
    1. Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
    2. Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
    3. Department of Pharmaceutical Nanotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
    4. Department of Biology, Payame Noor University, Ardabil, Iran
    5. Department of Pharmaceutical Nanotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
    6. Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran


  • Introduction: Gene therapy as a promising solution to treat a wide variety of diseases has attracted a great attention. The advent of endonuclease-based genome editing techniques such as Meganucleases (MNs), Zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and CRISPR (clustered regularly interspaced short palindromic repeats) has revolutionized biotechnology and gene therapy [1]. The effective CRISPR system with its advantages such as power, high accuracy, high specificity, high sensitivity, low cost, simplicity and multiplexity has become a popular approach in molecular biology research with various purposes such as diagnosis and treatment, among the genome editing techniques [2, 3]. CRISPR system has two classes, class I includes type I, III and IV systems and class II includes type II, IV and VI systems. The CRISPR system consists of an endonuclease called "Cas" and a guide RNA sequence "gRNA" to identify and bind to the target site [4]. This genome editing system has a broad range of applications including imaging, screening, base editing, gene targeting, RNA editing and more [5]. However, the delivery of its elements into the target cells and subsequently the target genomic area is an important and determining factor in the success of the CRISPR genome editing system. CRISPR systems can be delivered in three formats including Cas9/sgRNA-Plasmid, Cas9/sgRNA-mRNA and Cas9/sgRNA-RNP into target cells for genome editing. Types of delivery methods including physical delivery methods (electroporation, microinjection and hydrodynamic injection) and viral delivery methods (adenovirus, lentivirus and adeno-associated viruses) are commonly used to deliver gene constructs. Overall, physical delivery methods, despite all their advantages, face challenges such as damage to cells, need for special equipment, high cost, cellular totoxicity, and so on. Despite their advantages and high efficiency, viral delivery methods also face disadvantages such as triggering an immune response, the risk of being integrated into the host genome and so on. Recently, different groups using nanomaterial-based delivery systems have successfully delivered CRISPR system components into target cells and carried out desired genomic operations. In general, delivery systems based on nanotechnology have advantages such as easy synthesis, high efficiency, high load capacity, high specificity, applicability in vivo, low cost, suitable size etc [6, 7].
  • Methods: In writing this review article, we searched in various databases such as google scholar for keywords related to CRISPR and delivery methods, and selected the most suitable papers.
  • Results: Sun et al. were the first to be able to transfer the components of the CRISPR system into desired cells using a nanotechnology-based delivery system (DNA nanoclew). First, the DNA nanoclew was synthesized using the Roling Circle Amplification (RCA) method and then Cas9/sgRNA was loaded on it. Finally, they coated the DNA nanoclew-Cas9/sgRNA complex using polyethyleneimine (PEI) [8]. Mout et al. using arginine-functionalized gold nanoparticles were able to successfully deliver the Cas9/sgRNA ribonucleoprotein complex into target cells with 90% efficiency. Finally, by evaluating the efficiency of indel generation by this system, it was found that the efficiency of this system in indel generation in AAVS1 and PTEN genes in HeLa cells was 29 and 30%, respectively, and in PTEN gene in the HEK-293T and Raw 264.7 cells, the efficiency was 23 was 28%, respectively [9]. Ming et al. successfully introduced gene-modifying proteins, including Cre recombinase and Cas9/sgRNA, into human cells and rat brain, using bioreductible lipid nanoparticles. They synthesized 12 bioreducible lipids by adding primary or secondary amine groups and acrylate. It was observed that the negatively charged Cas9/sgRNA complex is capable of forming complex with bioreducible lipids for effective gene editing. The EGFP gene in HEK cells was selected as the target gene to evaluate the delivery efficiency of Cas9/sgRNA complex by bioreducible lipids. It was found that the established system leads to cleavage of the EGFP gene and induces the NHEJ repair system in the EGFP gene and ultimately leads to loss of cell fluorescence [10].
  • Conclusion: Nanotechnology-based delivery systems for the delivery of CRISPR system components can be considered as suitable alternatives to traditional delivery systems due to their advantages.
  • Keywords: CRISPR, Delivery, Gene editing, Nanomaterial