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

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

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

### **5.5. Meganucleases**

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‐ cleases also offer high specificity and precision; however, historically, they were only capable of tolerating minor variations in their recognition site sequence, decreasing the probability of an available meganuclease for each desired application. In recent years, investigators have begun customizing meganucleases to expand their targeting repertoire. Two main approaches have been taken: modifying the specificity of existing meganucleases, and/or developing chimeric meganucleases with new recognition sites. In the latter approach, by fusing the DNAbinding domains of two different meganucleases, functional heterodimers can be designed for optimal efficacy and specificity (Figure 4E).
