International Journal of Molecular Sciences Review Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors Karim Shalaby 1 , Mustapha Aouida 1,* and Omar El-Agnaf 1,2,* 1 Division of Biological and Biomedical Sciences (BBS), College of Health & Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar; [email protected] 2 Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar * Correspondence: [email protected] (M.A.); [email protected] (O.E.-A.) Received: 12 September 2020; Accepted: 27 September 2020; Published: 5 October 2020 Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes. Keywords: CRISPR-Cas; gene editing; gene therapy; non-viral vectors; cell-penetrating peptides 1. Introduction The CRISPR genome editing system was first identified as short repeats of DNA downstream of the iap gene of Escherichia coli [1]. In 2002, it was referred to as “CRISPR”, and the CRISPR-associated genes were discovered as highly conserved gene clusters located adjacent to the repeats [2]. A series of studies later revealed that CRISPR is a bacterial immune system that dismantles invading viral genetic material and integrates short segments of it within the array of repeated elements [3,4]. This array is transcribed and the transcript is cut into short CRISPR RNAs (crRNAs) each carrying a repeated element together with a spacer consisting of the viral DNA segment [5]. This crRNA guides a CRISPR-associated (Cas) nuclease towards invading viral DNA to cleave it and inactivate viral infection at the next occurrence [5]. Starting 2013, the fusion of a crRNA containing a guiding sequence with a trans-activating RNA (tracrRNA) bearing the repeat, into a single-guide RNA (sgRNA) has opened the doors for gene editing in mammalian cell lines and many other species [6–10]. The flexibility of the CRISPR system lies in its programmable Cas nuclease, which utilizes a guide RNA (gRNA) sequence to reach the desired complementary genomic sequence [11]. CRISPR-Cas systems open venues for applications in genetic functional screening, disease modeling, and gene modification [12]. The double-stranded breaks (DSBs) created by the Cas nuclease makes deletions or insertions at precise genomic loci possible [12]. In nature, DSBs can occur randomly Int. J. Mol. Sci. 2020, 21, 7353; doi:10.3390/ijms21197353 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7353 2 of 26 Int. J. Mol. Sci. 2020, 21, x 2 of 27 during replication or due to environmental factors and are repaired in our cells using the homologous or insertions at precise genomic loci possible [12]. In nature, DSBs can occur randomly during DNA copy as a template in a natural DSB DNA repair process called homology-directed repair replication or due to environmental factors and are repaired in our cells using the homologous DNA (HDR)copy [13 as]. a This template cellular in a DNAnatural repair DSB DNA pathway repair can process thus becalled employed homology-directed to copy a co-introduced repair (HDR) [13]. artificial DNAThis template cellular (donor) DNA repair carrying pathway a desired can thus sequence be employed into the to target copy cleavagea co-introduced site (Figure artificial1). However,DNA HDRtemplate is only activated(donor) carrying when thea desired homologous sequence sister into th chromatide target cleavage is available, site (Figure usually 1). However, in S and G2 HDR phases, duringis only the activated cell cycle when [13]. the Otherwise, homologous HDR sister is suppressedchromatid is andavailable, DSB repairusually is in maintained S and G2 phases, through a distinctduring repair the pathwaycell cycle called[13]. Otherwise, non-homologous HDR is suppressed end-joining and (NHEJ) DSB [repair13]. In is non-dividing maintained through cells, such a as neurons,distinct gene repair editing pathway is mostly called carriednon-homologous out using end-joining NHEJ, where (NHEJ) broken [13]. DNAIn non-dividing ends are directly cells, such ligated withoutas neurons, any requirement gene editing of is sequence mostly carried homology out using [13]. NHEJNHEJ, mediatedwhere broken repair, DNA however, ends are is directly error-prone andligated tends towithout produce any arbitraryrequirement mutations of sequence at thehomo sitelogy of [13]. ligation NHEJ of mediated DSBs. Often, repair, thesehowever, mutations is error-prone and tends to produce arbitrary mutations at the site of ligation of DSBs. Often, these create premature stop codons or frame-shifts that are capable of knocking-out the gene of interest, mutations create premature stop codons or frame-shifts that are capable of knocking-out the gene of thus providing means to investigate gene function, develop a disease model, or to cease the expression interest, thus providing means to investigate gene function, develop a disease model, or to cease the of a harmfulexpression mutant of a harmful protein muta fornt therapy protein [for12 ].therapy [12]. Figure 1. Gene editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Figure 1. Gene editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems relies on DNA repair pathways. DNA double-stranded breaks (DSBs) are repaired in cells systems relies on DNA repair pathways. DNA double-stranded breaks (DSBs) are repaired in cells via the error-prone non-homologous end-joining (NHEJ), or the error-free homologous recombination via the error-prone non-homologous end-joining (NHEJ), or the error-free homologous (HR), the most common form of homology-directed repair (HDR). The DSB repair through NHEJ recombination (HR), the most common form of homology-directed repair (HDR). The DSB repair createsthrough small NHEJ insertions creates or small deletions insertions (indels), or deletions while HDR (indels), requires while aHDR repair requires template, a repair which template, could be a sisterwhich chromatid, could be another a sister homologouschromatid, another region, homo or anlogous exogenous region, or repair an exogenous donor. repair donor. AlthoughAlthough CRISPR CRISPR is is not not the the first first site-specificsite-specific nucl nucleaseease used used in ingene-editing, gene-editing, it is itthe is simplest, the simplest, mostmost rapid, rapid, and and cheapest cheapest of of the the designer designer nucleases.nucleases. CRISPR-Cas CRISPR-Cas systems systems areare classified classified into into2 classes, 2 classes, 6 types6 types and and 33 subtypes, 33 subtypes, diff differingering in theirin their genes, genes, protein protein sub-units sub-units and and the the structure structure ofof theirtheir gRNAs [14]. Class[14]. 2 systems Class 2 systems have been have the been most the preferred most preferre for geneticd for genetic manipulation, manipulation, due due to their to their simple simple design solelydesign involving solely a involving sequence-specific a sequence-specific monomeric monomeric Cas nuclease Cas guidednuclease by guided a variable by a gRNA variable molecule gRNA [15]. DSBsmolecule are created [15]. byDSBs the are Cas created nuclease by the after Cas gRNAnuclease aligns after gRNA with thealigns target with DNA the target sequence DNA sequence via base pair (bp)via matching base pair [16 (bp)]. Thus, matching only [16]. simple Thus, modification only simple ofmodifi the gRNAcation of sequence the gRNA is enoughsequence to is targetenough di tofferent target different locations in the genome [12]. The availability of target sites is limited due to the locations in the genome [12]. The availability of target sites is limited due to the requirement of a requirement of a protospacer adjacent motif (PAM) sequence adjacent to the target sequence [16]. But protospacer adjacent motif (PAM) sequence adjacent to the target sequence [16]. But PAMs are highly PAMs are highly common and virtually any site in the genome can be targeted [17]. Class 2 CRISPR commonsystems and which virtually have any been site incommonly the genome employed can be targetedfor genome [17 ].editing Class 2in CRISPR mammalian systems cells which with have been commonly employed for genome editing