INNOVATIONS IN NON-VIRAL DELIVERY & TARGET SPECIFICITY OF CRISPR/Cas9 GENE EDITING TECHNOLOGY
The discovery of CRISPR/Cas nucleases in E.coli uncovered a gene superfamily that evolved in archaea and bacteria, as a form of acquired immunity to repeated virus infection and plasmid transfection [1; 2]. The adaptability of this system as a gene editing tool in eukaryotic cells [3; 4] highlighted the potential of CRISPR/Cas nucleases to modify the nucleic acid-based genome of any species at a single or at multiple loci, following its delivery to its target as DNA, mRNA, or as a ribonucleoprotein (RNP) complex. While these attributes have captured the collective imagination of scientists, CRISPR/Cas gene editing technology is not without its challenges. In the case of CRISPR/Cas9 nuclease, successful eukaryotic gene editing hinges not only on the choice of cell target and its pre-mutational genotype and phenotype, but also, on the following considerations:
- The choice of vector (i.e. virus- or non-virus), that will ensure efficient and targeted delivery of its payload, taking into account the context (i.e., in vitro or in vivo).
- The design and robust experimental validation of the gene editing components.
- The format of the CRISPR/Cas9 cargo that will be delivered to its target (i.e. nucleic acid or ribonucleoprotein (RNP)).
- The possibility that uncontrolled gene editing activity could occur within the intended target leading to immediate/ latent unintended effects of the mutation.
- The risks of dose- or vector- or gene-related oncogenesis, toxicity, cytotoxicity or immunogenicity.
- The risk that the CRISPR/Cas9 machinery is trapped/degraded within exosomes, preventing its delivery to its target locus (loci).
- The design of protocols for post-mutational analyses of genotype and phenotype.
- A strategy to identify phenotypes that can be attributed to off-target mutations.
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Here, we focus on two approaches to the safe and efficient design and delivery of CRISPR/Cas9 gene editing technology, namely:
The development of alternatives to virus-mediated delivery of CRISPR/Cas9, in vitro and in vivo, and Innovations that have been incorporated into the CRISPR/Cas9 machinery itself, to enhance its target specificity and efficacy.
1. Current and emerging alternatives to virus-mediated delivery of CRISPR/Cas9
There are many reports of successful disease treatment by virus-mediated delivery of modifying genes [6;7]. However, the popularity of integrating and non-integrating virus vectors has waned. This may be because (i) they do not deliver their genetic payload to their target reliably, (ii) there is an upper limit to the size of the nucleic acid cargo that can be inserted into the virus genome, (iii) large-scale production of virus vectors is difficult; and (iv) the virus itself carries a real risk of causing neoplastic transformation, immunogenicity, cytotoxicity or toxicity [5;8;9]. Several noteworthy alternatives to virus-mediated delivery of CRISPR/Cas9 are in development:
Applications intended for in vivo use include:
(i) lipid nanoparticle-mediated delivery of Cas9 mRNA combined with adeno-associated viruses (AAV) encoding a sgRNA and repair template , (ii) co-delivery of a Cas9 protein: sgRNA : lipofectamine complex that is seeded onto a polyDOPA-melanin scaffold, and is intended to provide localized and sustained editing in vivo, which could be useful in tissue regeneration , and (iii) Improved Genome editing via Oviductal Nucleic Acids Delivery, (i-GONAD) [11;12], which caters for knock-in, knock-out and large deletion mutations, and is a candidate for correction of germline mutations in vivo, because the CRISPR/Cas9 machinery is introduced into pre-implantation-stage embryos.
Applications intended for in vivo or in vitro use include:
(iv) co-delivery of Cas9 mRNA and sgRNA within tricarboxamide lipid-like nanoparticles , or (v) within zwitterionic amino lipid nanoparticles (ZNPs) , or (vi) co-delivery of Cas9 protein and sgRNA using an amphipathic “Endo-Porter” (EP) peptide .
In vitro only applications include:
(vii) co-delivery of Cas9 protein and sgRNA within exosome-liposome hybrid nanoparticles (viii) direct cytosolic delivery of Cas9 protein complexed with sgRNA in gold nanoparticles  CRISPR/Cas9 RNP-mediated gene editing by tube electroporation , and (x) co-microinjection of Cas9 mRNA and sgRNAs into 1 cell-stage embryos [19, 20].
2. Innovations to the CRISPR/Cas9 ribonucleoprotein machinery
Coupled with the development of non-virus vectors, are innovations in the design of the CRISPR/Cas9 complex that are intended to reduce both target non-specificity and sustained nuclease activity. First, is a “Kamikaze” CRISPR/Cas9 system , which uses an adeno-associated virus (AAV2) vector to co-deliver the Streptococcus pyogenes Cas9 gene (SpCas9) and sgRNAs in vivo, and to ensure that SpCas9 nuclease is degraded and the gene of interest is disrupted.
The second innovation is the development of a high-fidelity variant of SpCas9 that has been modified to reduce non-specific DNA contacts .
3. Concluding remarks
The innovations described above, are just some of the many recent attempts to advance CRISPR/Cas9 technology towards clinical translation. The aims are to reduce or eliminate the risks of unintended indels, sustained or curtailed expression of Cas9, dose-dependent toxicity, cytotoxicity, immunogenicity, exosomal entrapment, premature degradation of the CRISPR/Cas9 machinery, and to ensure that the cargo remains stable, effective and accessible to its target(s).
ISHINO, Y, SHINAGAWA, H, MAKINO, K, et al. “Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product”. J Bacteriol 1987; 169(12): 5429-5433.
MILLER, JB, ZHANG, S, KOS, P, et al. “Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA”. Angew Chem Int Ed Engl 2017; 56(4): 1059-1063.
SHEN, Y, COHEN, JL, NICOLORO, SM, et al. “CRISPR-delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure”. J Biol Chem 2018; 293: 17291-17305.