**6. The best vehicle for the job**

In genome editing, the identification of components capable of eliciting HDR is only half the battle. Efficiently delivering those components to the target cell can be an equally important hurdle to overcome. Unlike gene and transcript replacement therapies, where the goal is to achieve stable, long-term expression of a supplemental protein, gene editing has the advantage of requiring only short-term expression of the foreign components in the cell. Once these components have been expressed, induced a DSB, and triggered HDR, their presence is no longer necessary. In fact, in the attempt to minimize unwanted off-target cleavage activity and prolonged DSB induction, the ideal nuclease-delivery vehicle should only be transiently expressed.

Additionally, without the need for integration to promote stable expression, non-integrating delivery vehicles are also preferable, to minimize the risk of insertional mutagenesis. Delivery vehicles must also be capable of transducing target cells efficiently, to facilitate gene correction in enough cells to overcome the initial defect. To summarize, since transient expression of the nuclease is sufficient for stable modification of the genome, the ideal nuclease delivery vehicle should be:

**•** Short-lived,

combination then guides a Cas9 DNA nuclease to a specific location within the viral DNA,

Investigators discovered that by designing a new crRNA and combining it with the tracrRNA, a "single-guide RNA" (sgRNA) could be produced that would direct the Cas9 nuclease activity to any desired location. Studies have shown that delivery of two components, the Cas9 nuclease and a corresponding sgRNA (containing both the crRNA and tracrRNA), were sufficient to elicit cleavage in a desired gene [52–55]. Hence, by retargeting the crRNA portion

Unlike ZFN and TALEN strategies, the nuclease cleavage domain in the CRISPR/Cas9 system is not fused to the DNA binding domain: instead, these are delivered to the cell in two separate components (Figure 4C). As a result of this design, only a single DNA binding domain has to be created. As this single protein-binding domain is significantly shorter than those required for TALEN or ZFN designs, CRISPR/Cas9 components are significantly easier and more cost effective to synthesize, making this technology more widely available to the research com‐ munity at large. Despite lower costs and greater accessibility, the functional activity of CRISPR/ Cas systems appears to be equal to or greater than their ZFN and TALEN counterparts.

As the CRISPR/Cas9 system is a relatively new genome engineering technology, it will be important for the field to thoroughly study any potential shortcomings. For instance, since a relatively short DNA binding domain and cleavage site are utilized, the risk of low specificity

In an effort to reduce the risk of unwanted off-target mutations associated with monomeric CRISPR/Cas9 nucleases, a modified dimeric version has now been developed [57]. Where the monomeric Cas9 nuclease is recruited by one sgRNA of only ~100 nucleotides in length (with 17–20 nucleotides of complementarity to the target), dimerization offers an attractive strategy

In this approach, a wild-type *Fok*I nuclease domain is fused to a catalytically inactive Cas9 (dCas9) protein. Two such *Fok*I-dCas9 fusions are recruited by two corresponding guide RNAs, where both are required to bind their respective target sites in order for *Fok*I dimerization and DSB induction to occur (Figure 4D). An appropriately designed spacer and protospacer adjacent motif (PAM) are also critical for driving efficient cleavage. Overall, this RNA-guided *Fok*I nuclease (RFN) strategy has been shown to elicit robust genome editing efficiencies while

Meganucleases are another form of endonuclease utilized for genome editing approaches. They are unique in that their DNA recognition and cleavage functions are naturally combined in a single domain. There are five classes available, where I-SceI, I-CreI, and I-DmoI are perhaps the most widely used. Consisting of a large recognition site of 12 to 40 base pairs, meganu‐

of the sgRNA, a site-specific genome-editing tool could be developed (Figure 4C).

called the protospacer, where a DSB is induced.

368 Cystic Fibrosis in the Light of New Research

and potential off-target recognition may be greater [56].

for improving the binding specificity of the Cas9 system (Figure 4D).

reducing known off-target mutations to undetectable levels [57].

**5.5. Meganucleases**

**5.4. Dimeric CRISPR RNA-guided** *Fok***I nucleases**


A variety of vectors have been utilized to deliver genome-editing reagents to the cell. Initial *in vivo* studies have included AAV viral vectors as well as integrase-defective lentiviruses. Using AAV-encoded ZFNs, for instance, Li and colleagues demonstrated direct *in vivo* gene correction using HDR in a murine model of FIX deficiency [36]. Despite these successes, early *in vivo* strategies have not fulfilled two of the critical components for nuclease delivery vehicles: transience and lack of potential integration. In addition, with respect to translation of these approaches to CF airway disease, none of these strategies reported targeting cells in the lung.

Due to its short half-life and inability to integrate into the genome, modified mRNA is gaining interest as an ideal vector for site-specific nuclease delivery: one that would address two of the main outstanding issues previously discussed. Transient expression of mRNA-encoded nucleases would also minimize the long-term threat of off-target events associated with the use of stably expressing, and possibly integrating viral vectors.
