CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery

Abstract

Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications of gRNA. Finally, we consider the extracellular and intracellular barriers to nonviral CRISPR/Cas9 delivery and propose strategies that may overcome these barriers to realize the clinical potential of CRISPR/Cas9-based genome editing.

Tapping the RNA world for therapeutics

“…The same strategies that deliver siRNAs to the liver or elsewhere in the body can also be employed to deliver miRNA mimics, either as stem-loops or as double-stranded ~22-nucleotide modified RNAs that resemble the endogenous Dicer-cleaved miRNA 80,95 . The lessons learned about delivering siRNAs will probably facilitate the development of ways to deliver the larger cargo being contemplated for therapeutics using modified mRNAs 96,97 or CRISPR-Cas-mediated gene editing 98–104 . However, the delivery obstacles for these approaches are greater than those for siRNAs and should not be underestimated.…”

“…This includes activation and suppression of gene expression and genome-wide screening 98–104 . Like RNAi, the specificity of CRISPR-Cas relies on the antisense pairing of sgRNAs to specific genes, but on chromosomal DNA rather than RNA (Fig.…”

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…However, base editors are large fusion proteins that exceed the packaging capacity of AAV. Further efforts to reduce the size (Friedland et al, 2015; Zetsche et al, 2015b) and limit immunogenicity of base editing components or vehicles (Abudayyeh et al, 2016; Bessis et al, 2004; Chew et al, 2016; Hirsch et al, 2016; Wang et al, 2017; Yin et al, 2016; Zuris et al, 2015) may also improve their clinical utility.…”

CRISPR genome editing and its medical applications

“…In fact, two lentiviruses linked to each other were used to express the CRISPR components in hematopoietic stem and progenitor cells (HSPC) in mice to change five genes ex vivo. Lack-offunction mutations generated in these cells led to acute myeloid leukaemia (AML) model design [48][49][50].…”