PROGRESS REPORT Gene Editing www.advtherap.com Synthetic Vehicles for Encapsulation and Delivery of CRISPR/Cas9 Gene Editing Machinery Valentina Carboni, Carine Maaliki, Mram Alyami, Shahad Alsaiari, and Niveen Khashab* Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated 9 (Cas-9) technology holds tremendous potential as a gene editing tool. Different strategies have been adopted for in vitro and in vivo delivery of CRISPR/Cas9, including both viral and non-viral. The possibility of tailoring properties of nanosized systems makes the molecular design of self-assembled non-viral delivery systems based on organic (lipids and polymers) and hybrid (zeolitic imidazolate frameworks, ZIF and gold nanoparticles) materials of a great interest in CRISPR/Cas9 delivery. This review highlights the progress and challenges of organic and hybrid CRISPR/Cas9 delivery vehicles. 1. Introduction The CRISPR/Cas9, clustered regularly interspaced short palin- dromic repeat (CRISPR) and CRISPR-associated 9 (Cas9) tech- nology, provides an efficient means of introducing targeted loss function mutations at specific sites in the genome. [1] The gene editing method requires Cas 9 nuclease and an engineered single guide ribonucleic acid (RNA) (sgRNA). sgRNA within the protein complex targets the deoxyribonucleic acid (DNA) sequence of in- terest; subsequently, both DNA strands are cleaved by the Cas9 protein. The break is then repaired by non-homologous end join- ing (NHEJ) or homology-directed repair (HR). The simplicity of designing CRISPR/Cas9 to edit or alter specific genomic loci suggests a new way to interrogate gene function on genome wide scale and repair genetic defects. [2] CRISPR/Cas9 showed a great potential in gene editing and this gene editing machinery can be delivered into cells in three dif- ferent ways: 1) DNA plasmid that encodes both the Cas9 protein and the guide RNA, 2) messenger RNA (mRNA) for Cas9 transla- tion and a separated single guide RNA, 3) ribonucleoprotein com- plex, composed of the Cas9 protein and guide RNA. This last rep- resents the most straightforward delivery approach, since it by- passes additional transcription and/or translation passages and offers the most transient functionality of genome editing with Dr. V. Carboni, Dr. C. Maaliki, M. Alyami, Dr. S. Alsaiari, Prof. N. Khashab Smart Hybrid Materials Laboratory (SHMs), Advanced Membranes and Porous Materials Center King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia E-mail: niveen.khashab@kaust.edu.sa The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adtp.201800085 DOI: 10.1002/adtp.201800085 reduced off-target effects and toxicity. How- ever, the large size of the Cas9 protein, the positive charge of Cas9 protein, and the strong negative charge of guide RNA make their delivery quite difficult and their uptake is still limited by a number of physiologi- cal barriers. Therefore, previously existing gene transfection approaches were used to deliver CRISPR/Cas9 plasmid, [3] leading to more customized, efficient, and innovative delivery platforms. [4] Considering the nature of delivery vectors, [5] CRISPR/Cas delivery platforms are divided into viral [6] and nonviral vectors. [7] Although viral vectors are more efficient in delivery, they are highly immunogenic. Nonviral vectors are less immuno- genic and they can be labeled as physical or chemical, according to the nature of the process of the carriers involved. Physical methods enable cell transfection by creating transient defects in the phospholipid bilayer of cell membrane or by di- rect injection and they represent one option for CRISPR/Cas9 complex delivery. The simplest technique is microinjection: [8] it consists of the direct injection of CRISPR/Cas9 and this can be achieved at the cellular level by using micro-scale needles. It is a very efficient transfection technique, since it allows precise de- livery into cytoplasm or nucleus, thus directly bypassing all the extracellular barriers that usually hamper efficient delivery. How- ever, it requires manual injection on each cell of the targeted and this is translated in high level of sophistication and long timing for sample preparation, making this approach impractical for ex- periments in which a large number of cells are involved. One of the most used method (both in vitro and in vivo) is electroporation, [9] that refers to an increased cell membrane per- meability toward biomolecules as a consequence of the electrical field applied onto cell membrane. This method proved to be very efficient and safe, but it suffers from several disadvantages, such as irreversible changes of cell membrane physiology, loss of cell mobility, and cell death. Pressure can be an alternative trigger for CRISPR/Cas9 inter- nalization in cells in vivo: this approach refers to hydrodynamic injection, [10] in which a liquid solution is injected intravenously at high volume and pressure, ensuring enhanced cell membrane permeability. This method requires large injection volumes (up to 10% of body weight) and it is mainly limited by gene delivery to liver in rodents. Other approaches involve mechanical deformation, [11] sonoporation, [12] laser irradiation, [13] and osmocytosis, [14] but these methods can damage cells. On the other hand, chemical methods represent a very promis- ing approach for in vivo delivery of CRISPR/Cas9 complex, due Adv. Therap. 2019, 2, 1800085 C  2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1800085 (1 of 9)