**4. Conclusion**

and isolated by fluorescence-activated cell sorting (FACS) with fluorescence labeled antigen followed by iterative rounds of SHM to yield high affinity antibodies [34]. In mammalian, yeast and bacterial cell surface display it is essential to select and isolate the cells that are able to produce functional antibodies. Cell-based sorting such as high throughput flow cytometry, allows high throughput screening of cells per minute, analyzing the cells according to size, granularity and binding to fluorescence labeled antigens. The utmost reason for antibody engineering is the production of human monoclonal antibody in large scale, hence, it is crucial to implement a selection towards antibody expressing mammalian cells. Mammalian display allows for screening of eukaryotes-expressed full length antibod-

The advancement of genome editing technologies offers a new approach to create sequence diversity. In fact, cells can repair DNA damages intrinsically by joining two ends together or filling the gap with similar sequences. However, cells can also repair the break by using a new piece of DNA that has the desired mutation. This is the basis of *in vivo* genome editing technologies today [124]. Extensive functional genomics studies helps to provide the insights required that targeted DNA double-stranded breaks (DSBs). The DSBs can induce genome editing via homologous recombination (HR) in the presence of exogenous homology repair template, as well as error-prone non-homologous end-joining repair (NHEJ) pathway in the absence of repair template. These two pathways are versatile to allow precise genome modification [125]. To date, there are four major classes of engineered DNA binding proteins to target DSBs: meganucleases, zinc finger (ZF) nucleases, transcription activator-like effectors

Meganucleases are derived from microbial mobile genetic elements that integrate nucle-

eukaryotic transcription factors that contain the DNA binding domain and is similar to a set of three fingers, with each finger contacting with 3 nucleotides of DNA [126]. TALEs are produced naturally by *Xanthomonas* sp. that consist of DNA binding domains with 30–35 tandem repeats, with each domain recognizing a single nucleotide of DNA. Specificity of TALEs is governed by the two amino acids that are known as the repeat-hypervariable diresidues [127]. Both ZF nucleases and TALEs require *Fok*I nuclease to direct the nucleolytic activity towards the genome locus for modifications. Recently, RNA-guided DNA endonuclease Cas9 is given much more attention due to its simplicity and versatile approach towards *in vivo* genome engineering. It is derived from type II bacterial adaptive immune system. The CRISPR-Cas9 mediated immunity is explicitly explained in Yang et al. [128]. The hallmark of this system is the short RNA guide that recognizes the target DNA through base pairing, forming a RNA-DNA complex, subsequently Cas9 creates DSB on the target sequence [129,

While other approaches have their own limitations, the robustness of the CRISPR-Cas9 system sheds some light on direct endogenous genome editing on virtually any organism of choice. Meganuclease lacks target specificity, which is why it is not widely employed. However, ZF domains have a tendency to crosslink with neighboring protein -domains or -complexes

His2

bound to a single atom of zinc) are

ies with correct glycosylation [18, 123].

34 Antibody Engineering

(TALEs) and RNA-guided DNA endonuclease Cas9 [124].

130] and a designed DNA fragment can be specifically incorporated.

ase and DNA binding domains. ZF nucleases (Cys<sup>2</sup>

Naïve and synthetic human antibody repertoires are a very valuable source for the selection of antibodies against nearly any antigen. The role display technologies play in the quest to generate monoclonal antibodies from these libraries is obvious with the increasing number of antibody lead candidates going into clinical trials.

Affinity maturation of selected binders is now possible by expressing for example the AID enzyme during selection of antibodies using antibody mammalian cell surface display or by using a pool of microchip-synthesized CDRs incorporated into an antibody framework. Selection of naïve and synthetic recombinant antibodies combined with *in vitro* and *in vivo* affinity maturation techniques will have a profound effect on the generation of high affinity diagnostic and therapeutic human antibodies.
