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Engineering Nucleic Acid Chemistry for Precise and Controllable CRISPR

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Engineering Nucleic Acid Chemistry for Precise and Controllable CRISPR Science Bulletin 64 (2019) 1841–1849 Contents lists available at ScienceDirect Science Bulletin journal homepage: www.elsevier.com/locate/scib Review Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing ⇑ Weiqi Cai a,b, Ming Wang a,b, a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecule Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b University of Chinese Academy of Sciences, Beijing 100049, China article info abstract Article history: The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/ Received 21 June 2019 Cas9) genome editing technology is revolutionizing our approach and capability to precisely manipulate Received in revised form 16 July 2019 the genetic flow of mammalians. The facile programmability of Cas9 protein and guide RNA (gRNA) Accepted 23 July 2019 sequence has recently expanded biomedical application of CRISPR/Cas9 technology from editing mam- Available online 30 July 2019 malian genome to various genetic manipulations. The therapeutic and clinical translation potential of CRISPR/Cas9 genome editing, however, are challenged by its off-target effect and low genome editing effi- Keywords: ciency. In this regard, developing new Cas9 variants and conditional control of Cas9/gRNA activity are of CRISPR/Cas9 genome editing great potential for improving genome editing accuracy and on-target efficiency. In this review, we sum- RNA engineering Nucleic acid chemistry marize chemical strategies that have been developed recently to engineer the nucleic acid chemistry of Gene therapy gRNA to enhance CRISPR/Cas9 genome editing efficacy, specificity and controllability. This review aims to Aptamer highlight the endeavor that has been made to solve bottleneck problems in the field of CRISPR/Cas9 and inspire innovative researches to fulfill the gap between bench and bed. Ó 2019 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved. 1. Introduction CRISPR/Cas9 simply requires control of a short sequence (usually 20 nt) of gRNA to target gene loci. Based on its modularity and flex- Over past decades, the rapid development of gene editing tool- ibility, orthogonal and multiplexed gene editing can be easily sets has invigorated gene therapy, among which CRISPR/Cas9 achieved by simply customizing gRNA sequences [5]. In recent (Clustered Regularly Interspaced Short Palindromic Repeat and years, increasing number of nucleases in CRISPR family are discov- CRISPR-associated protein 9) is the top hit [1,2]. CRISPR/Cas9 gen- ered [6] and characterized [7,8], imparting researchers with ome editing is adapted from an immune defense system presents diverse tools to better elucidate and manipulate gene function. in many bacteria and archaea to protect them from the invasion The easy-to-handle and programable properties of CRISPR/Cas9 of bacteriophages. It is composed of Cas9 protein, an endonuclease, have led to generation of extensive biomedical applications [9], and guide RNA (gRNA) [3] that anchoring Cas9 to the targeting including gene therapy [10], nucleic acid detection [11] and gen- gene loci flanked by a protospacer-adjacent motif (PAM) (Fig. 1a). ome imaging [12]. Catalytically inactive Cas9 (dCas9) and Nickase In the presence of gRNA, Cas9 protein creates double strand breaks are also instrumental recently in transcriptional regulation [13,14], (DSB) which are subsequently repaired via two different pathways base editing [15], epigenetic editing [16], etc. (Fig. 1b), non-homologous end joining (NHEJ) or homology- As a promising therapeutic agent candidate, safety of CRISPR/ directed repair (HDR) inside cells, enabling a precise manipulation Cas9 genome editing is of most concern. Chances are CRISPR/ of genetic information of mammalians [4]. Cas9 induces imprecise genomic alteration or edits at unintended Unlike traditional genome editing systems, such as ZFN (zinc- loci because mismatch of gRNA is tolerated to a certain extent finger nucleases) or TALEN (transcription activator-like effector [17]. Due to the dynamic inferior of off-target activity to on- nucleases) relying on engineering arrays of DNA-binding domains target activity, off-target activity can be repressed by restricting to recognize and target genome sites via protein-DNA interaction, Cas9 protein to a brief window [18] or mutating it into a high- fidelity variant at an expense of on-target editing activity [19,20]. Paired Cas9 Nickases have been utilized to increase their targeting SPECIAL ISSUE: Emerging Investigators 2019 specificity [21,22], but they incur extra potential off-target sites ⇑ Corresponding author. and make in vivo delivery [23] even more troublesome. E-mail address: [email protected] (M. Wang). https://doi.org/10.1016/j.scib.2019.07.035 2095-9273/Ó 2019 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved. 1842 W. Cai, M. Wang / Science Bulletin 64 (2019) 1841–1849 Fig. 1. (Color online) (a) CRISPR/Cas9 is a RNA guided nuclease adapted for specific gene editing. After positioned by guide RNA (gRNA) in the target DNA sequence downstream of a protospacer-adjacent motif (PAM), HNH domain of Cas9 protein generates a break at target DNA strand and RuvC domain breaks nontarget DNA strand. (b) DNA double-strand breaks (DSB) induced by CRISPR/Cas9 can be repaired via different repair pathways. Non-homologous end joining (NHEJ) is error-prone and creates insertion or deletion mutations (indels) at DSB. In assistance of donor DNA templates, the homology-directed repair (HDR) pathway provides a way to knock in gene fragments. Reprinted with permission from Ref. [4]. Copyright 2014 Springer Nature. As tight time, space and dose control over CRISPR/Cas9 is highly are tolerant to mutant, bulge and nexus are conserved and neces- desirable, scientists have been seeking for approaches to equip sary for DNA cleavage. The nexus and the hairpins interact with CRISPR/Cas9 with molecular safeguards. To date, most repurposed CRISPR/Cas9 systems focus on Cas9 protein engineering. For exam- ple, fuse Cas9 protein with other functional domains [24] and incorporate light-responsive unnatural amino acid site specifically [25]. However, inherent limitations have stymied engineering CRISPR/Cas9 through protein regulation. Despite diversity of pro- tein engineering processes [26], they all require sophisticated and burdensome gene manipulation or directed evolution. Further- more, as Cas9 variants and other CRISPR/Cas9 system keep flour- ishing, these strategies have to be reinvented from the very beginning. An alternative way of controllable CRISPR/Cas9 genome editing is to optimize gRNA to modulate its recognition of gene loci. The most widely applied Cas9 is furnished with CRISPR RNAs (crRNAs) and trans-activating crRNA (tracrRNA) or a chimeric version ter- med single guide RNA (sgRNA) [3]. The crRNA recognizes and hybridizes with matching DNA, forming a R-loop structure which plays a key role in DNA cleavage [27]. The tracrRNA partially com- plements crRNA and is responsible for maintaining Cas9 protein conformation in active state [28]. Adopting functions of natural dual crRNA-tracrRNA duplex (Fig. 2a), sgRNA mimics their struc- ture and can be easily produced from in vitro transcription. The sgRNA (Fig. 2b) is constituted by six conserved modules: the Fig. 2. (Color online) (a) Schematic representation of crRNA (blue) and tracrRNA spacer, the lower stem, the upper stem, the bulge, the nexus and (red). (b) Cas9 sgRNA includes six functional modules: spacer responsible for DNA targeting (black); the upper stem (blue), bulge (orange), and lower stem (green) the hairpins [29]. The spacer forms RNA:DNA duplex when formed by the crRNA: tracrRNA duplex and the nexus (red) and hairpins (purple). engaged with genome. While the lower stem and the upper stem Reprinted with permission from Ref. [29]. Copyright 2014 Elsevier. W. Cai, M. Wang / Science Bulletin 64 (2019) 1841–1849 1843 Cas9 protein and define orthogonality of CRISPR/Cas system. Cas9 Lee et al. [46] went towards another direction of sgRNA ligation. recognizes a G-rich PAM at 30 end of protospacer and creates blunt They investigated the tolerance of gRNA to chemical modifications DSB. In contrast to Cas9 protein, gRNA adopts simple structures such as amide, azide, disulfide and DBCO. Introducing modification which can be predicted and depicted by abundant software tools. at 50 end of Cas9 crRNA will not compromise their activity. So they Deep insight into gRNA interaction [30] and large-scale profile directly conjugated crRNA with chemically modified donor DNA [31,32] permit efficient gRNA design with minimal off-target template and electroporated the complex to BFP-HEK293T or effect. Straightforward interaction between gRNA and DNA enables BFP-K562 cells together with Cas9 protein. The edited cells under- various engineering approaches to modulate gRNA structure to went DSB generation and HDR gene insertion with a higher effi- improve genome editing. Truncation is believed to decrease excess ciency than traditional methods. By labelling donor DNA with binding energy of the Cas9-sgRNA:genome complex, rendering fluorophore Alexa 647, the authors developed an easy approach lower tolerance of mismatches [33]. In the same vein, gRNA that to sort and enrich gene-edited cells using flow cytometry analysis. sterically impede
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