Current updates of CRISPR/Cas9-mediated genome editing and targeting within tumor cells: an innovative strategy of cancer management

Abstract

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9), an adaptive microbial immune system, has been exploited as a robust, accurate, efficient and programmable method for genome targeting and editing. This innovative and revolutionary technique can play a significant role in animal modeling, in vivo genome therapy, engineered cell therapy, cancer diagnosis and treatment. The CRISPR/Cas9 endonuclease system targets a specific genomic locus by single guide RNA (sgRNA), forming a heteroduplex with target DNA. The Streptococcus pyogenes Cas9/sgRNA:DNA complex reveals a bilobed architecture with target recognition and nuclease lobes. CRISPR/Cas9 assembly can be hijacked, and its nanoformulation can be engineered as a delivery system for different clinical utilizations. However, the efficient and safe delivery of the CRISPR/Cas9 system to target tissues and cancer cells is very challenging, limiting its clinical utilization. Viral delivery strategies of this system may have many advantages, but disadvantages such as immune system stimulation, tumor promotion risk and small insertion size outweigh these advantages. Thus, there is a desperate need to develop an efficient non-viral physical delivery system based on simple nanoformulations. The delivery strategies of CRISPR/Cas9 by a nanoparticle-based system have shown tremendous potential, such as easy and large-scale production, combination therapy, large insertion size and efficient in vivo applications. This review aims to provide in-depth updates on Streptococcus pyogenic CRISPR/Cas9 structure and its mechanistic understanding. In addition, the advances in its nanoformulation-based delivery systems, including lipid-based, polymeric structures and rigid NPs coupled to special ligands such as aptamers, TAT peptides and cell-penetrating peptides, are discussed. Furthermore, the clinical applications in different cancers, clinical trials and future prospects of CRISPR/Cas9 delivery and genome targeting are also discussed.

Keywords: CRISPR/Cas9; cancer; clinical trials; genome editing; mechanism; nanoparticles; structure.

FIGURE 1

Overall three‐dimensional structure of Streptococcus pyogenes Cas9‐sgRNA‐DNA ternary complex. (A) Domain organization. (B) Ribbon representation of Cas9‐sgRNA‐DNA complex at different angles, obtained from the protein data bank (https://www.rcsb.org, PDB ID: 4OO8), edited using software developed by Resource for Biocomputing, Visualization, and Informatics at University of California, San Francisco Chimera. Abbreviations: NUC, nuclease; REC, recognition; PI, PAM interacting; PAM, protospacer adjacent motif; sgRNA, single‐guide RNA

FIGURE 2

Biology of the CRISPR/Cas9 system and relevant transcription/translation products. Engineered CRISPR/Cas9 system can be devised for site‐specific genome editing as sgRNA:Cas9. Abbreviations: Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeats; tracrRNA, trans‐activating CRISPR RNA; Rec, recognition; Nuc, nuclease; sgRNA, single‐guide RNA

FIGURE 3

The secondary structure of sgRNA complexed with the target DNA. (A) sgRNA showing extra repeat‐antirepeat regions usually truncated in designing sgRNAs for genomic engineering. (B) Ribbon representation of sgRNA‐DNA complex. Abbreviations: crRNA, CRISPR RNA; tracrRNA, trans‐activating CRISPR RNA

FIGURE 4

Formation of a DSB and repair by NHEJ and HDR. Repair by NHEJ results in the formation of random indels. Repair by HDR requires a template DNA strand for precise repair. Abbreviations: DSB, double‐stranded break; HDR, homology‐directed repair; NHEJ, non‐homologous end joining

FIGURE 5

A proposed mechanism of target DNA recognition and its cleavage by the CRISPR/Cas9 system. (A) Large conformational rearrangement occurs in Cas9 upon sgRNA loading to achieve a target‐recognition mode. Apo‐Cas9 consists of a PAM‐interacting cleft, largely disordered, that becomes prestructured for PAM sampling. (B) The guide RNA seed is preorganized for interrogation of adjacent DNA for guide RNA complementarity. (C) A coordinated multiple steps further activate Cas9, starting with PAM recognistion. (D) Local DNA melting, RNA strand invasion and (E) subsequent R‐loop formation. (F) A conformational change of the HNH domain allosterically regulates the RuC domain, ensuring concerted DNA cleavage. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; Rec, recognition; Nuc, nuclease; sgRNA, single‐guide RNA; PAM, protospacer adjacent motif

FIGURE 6

Different strategies of CRISPR/Cas9 delivery as mRNA, DNA, or protein. These methods include adenovirus transport, electroporation, microinjection, and the use of multifunctional NPs. Abbreviations: Cas9/sgRNA, CRISPR associated protein 9/single‐guide RNA; CRISPR, clustered regularly interspaced short palindromic sequence; NPs, nanoparticles; NPC, nuclear pore complex