**5.2. Transcription activator-like effector nucleases**

Transcription activator-like effector (TAL effector, or TALE) proteins are secreted by *Xantho‐ monas* bacteria upon infecting various species of plant. They function by binding to promoter sequences in the host to upregulate plant genes that are beneficial to bacterial infection. Similar to the concept of ZFNs, TALENs are built by fusing the DNA binding domain of a TAL effector to a DNA cleavage domain with nuclease activity [44–47]. The DNA binding domain consists of multiple repeats of a 33-34 amino acid sequence, where all but the 12th and 13th amino acids are highly conserved. By selecting a combination of repeat segments with the appropriate variable regions (Repeat Variable Diresidues, or RVDs), specific DNA binding domains can be engineered.

TALENs utilize the same non-specific DNA cleavage domain from the *Fok*I endonuclease to confer cleavage activity. As a result, this strategy also requires two TALENs to work together as a pair, binding non-palindromic sequences on complementary strands of DNA. Proper positioning of the DNA binding and cleavage domains around the cute site allows the *Fok*I endonuclease domains to dimerize and produce a site-specific DSB (Figure 4B) [48,49].

Compared with ZFNs, TALENs can cleave a broader, more comprehensive range of DNA sequences. In addition, they tend to be more accurate, reducing the potential for off-target cleavage events. Furthermore, as a result of the fast ligation-based automatable solid-phase high-throughput (FLASH) system reported in 2012, large-scale assembly of TALENs has also become a more efficient and cost-effective alternative [50].

### **5.3. CRISPR/Cas9**

ZFNs utilize the non-specific DNA cleavage domain from the *Fok*I restriction endonuclease to confer their cleavage activity [39]. Their design traditionally incorporates the wild-type *Fok*I cleavage domain; however, more recent studies have utilized variants with improved cleavage activity or specificity [40–42]. Functioning as a dimer, the *Fok*I domain requires two ZFN constructs, working together as a pair: one ZFN binds to a sequence immediately upstream of the intended cut site, while the other targets sequence immediately downstream of the cut site on the complementary strand (Figure 4A). This alignment places the C-terminal nucleases at a desired distance apart across the cut site, where they dimerize and create a DSB. Proper

**Figure 4. Genome-editing nucleases: Mechanisms of action.** An overview of the mechanisms of DNA binding and cleavage used by ZFNs (**A**), TALENs (**B**), CRISPR/Cas9 (**C**), the dimeric CRISPR RNA-guided *Fok*I system (**D**), and meganucleases (**E**). PAM, protospacer adjacent motif; sgRNA, single guide RNA; RuvC, nuclease domain in Cas9 that cleaves the non-target strand of DNA; HNH, nuclease domain in Cas9 that cleaves the target strand of DNA.

Numerous evidence has supported the use of ZFNs for targeted gene editing in multiple species, including mice, rats, rabbits, pigs, plants, and zebrafish [38]. The use of this platform has also extended to the manipulation of stem cell populations *ex vivo*. In one seminal study, ZFNs facilitated targeted disruption of *CCR5*, a co-receptor involved in HIV entry [32]. By introducing the *CCR5*-∆32 mutation into *ex vivo* expanded CD4+ T cells, followed by engraft‐ ment into HIV-1 infected mice, these target cells no longer expressed functional CCR5 entry receptors, making them more resistant to infection. Further studies utilized a dual strategy to target both of the HIV entry co-receptors, CCR5 and CXCR4 [43]. The use of ZFNs was also

spacing of binding and cleavage domains is critical for optimal DSB induction.

366 Cystic Fibrosis in the Light of New Research

In 2012, the use of a novel genome-editing tool was described in human cell culture [51]. In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) work together with Cas genes to form a prokaryotic adaptive immune system that protects against foreign genetic elements such as plasmids or phages. Upon detecting viral DNA, for instance, this system converts segments of the foreign DNA into CRISPR RNAs (crRNA); the crRNA then combines with a trans-activating crRNA (tracrRNA). The crRNA–tracrRNA combination then guides a Cas9 DNA nuclease to a specific location within the viral DNA, called the protospacer, where a DSB is induced.

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

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