Engineering the Delivery System for CRISPR-based Genome Editing

By Z. Glass, Matthew L. Lee, Yamin Li, Qiaobing Xu. Trends in Biotechnology, 2018

299 citations recorded by the source corpus.

CRISPR-Cas systems, found in nature as microbial adaptive immune systems, have been repurposed into an important tool in biological engineering and genome editing, providing a programmable platform for precision gene targeting. These tools have immense promise as therapeutics that could potentially correct disease-causing mutations. However, CRISPR-Cas gene editing components must be transported directly to the nucleus of targeted cells to exert a therapeutic effect. Thus, efficient methods of delivery will be critical to the success of therapeutic genome editing applications. Here, we review current strategies available for in vivo delivery of CRISPR-Cas gene editing components and outline challenges that need to be addressed before this powerful tool can be deployed in the clinic. Next generation genome editing The development of genome editing tools has made great progress in the past decade. Originally limited to certain model organisms and inefficient at best, genome editing began to become more broadly applicable with the discovery of several programmable DNA binding proteins. Chief among these were zinc-finger (ZF) and transcription activator-like effectors (TALEs). By fusing these proteins to nucleases, it is possible to create a double-strand break at a specified site in the genome, which would then be repaired either through non-homologous end joining, usually leading to a small insertion or deletion and thus gene knock-out, or homologous recombination with a template, leading to insertion of a desired sequence of DNA [1, 2]. In practice, however, both ZFs and TALEs are difficult to reprogram, requiring extensive protein engineering for each new target. In early 2013, genome editing made a huge leap forward with the report that the microbial CRISPR – Cas9 (clustered regularly spaced palindromic repeat – CRISPR associated protein 9) adaptive immune system could be repurposed for genome editing in mammalian cells [3–5]. Unlike ZFs and TALEs, Cas9 uses a complementary RNA to achieve sequence specificity [6]. Moreover, Cas9 is a nuclease, obviating the need to fuse it to an additional protein. Cas9 can be programmed to target nearly any gene in the genome by synthesizing a guide RNA (gRNA) molecule which is complementary to the target sequence (Box 1) [7]. The design flexibility of the CRISPR system has driven the rapid adoption of this method for an array of genome editing applications in the laboratory [8]. Recently, as the alternative CRISPR nuclease Cpf1 has been discovered [9]. CRISPR-Cpf1 acts on a similar principle to CRISPR-Cas9, and the two techniques share many of the same advantages and disadvantages (Box 2). Even though CRISPR has already dramatically changed how molecular biology is conducted on the benchtop, some argue its true potential lies as a therapeutic. CRISPR, as well as ZFs and TALEs, offer the tantalizing possibility of treating or even eradicating thousands of human diseases at the level of DNA. By simple changing the gRNA and template DNA, Cas9 could go from repairing a mutation in CFTR in a patient with cystic fibrosis to knocking out an oncogene in a tumor of a cancer patient. However, like all genetic tools, Cas9-gRNA complexes must be delivered directly to the target cells’ nuclei to be effective. This challenge necessitates a particular focus on the delivery strategies and techniques in order to maximize the success rate and safety of this promising technology. Ultimately, successful delivery will allow for the treatment of a wide range of genetic disease and disorders in a clinical setting.

Subfields: Business and International Management, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/e6a8ef19e32ec612dca8809ffece26acf09fac25

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