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The use of DSBs to promote homologous recombination in mammalian cells was limited by the lack of a modular site‐specific endonuclease technology that could be directed to any gene of interest. The solution to this problem came from the work of Chandrasegaran´s group where for the first time the engineering of Zinc Finger‐based endonucleases (ZFN) was described (Kim et al. 1996). This system combines the modular design of Zinc Finger Domain to the powerful nuclease activity of the restriction enzyme FokI that requires dimerization to induce DSBs. The requirement of an obligate dimerization of FokI for an efficient cleavage guarantees high specificity to the system. The Zinc Finger DNA‐binding domain was selected based on the previous work from the labs of Klug and Pabo directed to the discovery of ZF domain (Miller et al. 1985) and the understanding of its structure (Pavletich and Pabo 1991).

Following the publication of the ZFN architecture, Carroll´s group showed for the first time efficient gene targeting using ZFNs in the Drosophila genome (Bibikova et al. 2002). This experiment not only resulted in efficient targeting by installation of random indels via NHEJ but it also demonstrated that ZFNs were precise enough to induce a number of DSBs in the genome compatible with embryonic development. In follow‐up works, gene insertion by Homology Directed Repair after ZFN cleavage was demonstrated by Carroll and Baltimore/Porteus groups, respectively, in Drosophila (Bibikova et al. 2001) and human cells (Porteus and Baltimore 2003).

The scene was set for preclinical genome editing, that is the demonstration of gene editing at disease‐relevant targets. The work from Fyodor Urnov and colleagues at Sangamo therapeutics to target the X‐linked (SCID) mutation in the IL2Rγ led to a new era in genome engineering (Urnov et al. 2005). The other important message coming from this work was the demonstration of bi‐allelic genome editing using targeted ZFNs, something that is very infrequent with classical gene targeting strategies as discussed above.

After the demonstration of preclinical genome editing, an important advancement in the field was the development of systems to engineer targeted endonucleases. These protein engineering pipelines resulted in the generation of precise endonucleases that could safely target the human genome without inducing strong toxicity as shown in the work of Joung, Kim, Cathomen & Voytas´ groups (Maeder et al. 2008; Kim et al. 2009).

It resulted evident from these early experiments that NHEJ is the main pathway of repair exploited by mammalian cells to repair DSBs. Based on these observations, most of the initial preclinical genome editing approaches were directed to exploit NHEJ for the generation of loss of function alleles. Scientists at Sangamo Therapeutics in collaboration with June´s group suggested that CCR5 could be an ideal target for the NHEJ‐dependent loss of function approach. This strategy was based on genomics data associating CCR5 loss of function to protection from HIV infection. Their work led to the first use of genome editing in primary human cells with efficient and safe genome editing (Perez et al. 2008; Maier et al. 2013).

The application of the NHEJ‐based loss of function strategy has also been important in biopharmaceuticals industry for the generation of modified CHO (Chinese Hamster Ovary cells) that are important in biopharmaceutical drug development (Santiago et al. 2008).

NHEJ‐dependent knock out was also reproduced in embryos to generate Knock Out in Drosophila, Rats, Mice (Geurts et al. 2009; Carbery et al. 2010) and Zebrafish (Doyon et al. 2008). This was another important milestone in Pharmaceutical industry because it allowed to validate drug targets in different organisms beyond mice bypassing the need for stem cell manipulation and at the same time speeding up the process to generate animal model of disease. These experiments changed the concept of model organism itself. Model organism can be potentially any animal model with a sequenced genome and where it is possible to deliver gene editors. This is exemplified by the generation of transgenic models using mRNA injection or electroporation in embryos bypassing the long process required for the development of transgenic models using stem cell manipulation.

Despite the initial successes to target mammalian genomes with ZFNs, the design of these enzymes was challenging and the technology was inaccessible to most labs. The advent of TALENs, published for the first time in 2009 (Boch et al. 2009; Moscou and Bogdanove 2009), started the democratization of the genome editing field. Also thanks to improvement of TALEN design by the laboratories of Joung (Reyon et al. 2012) and Voytas (Cermak et al. 2011), it was possible to design tailored targeted nucleases for potentially any gene and for any region of interest. This technology applied to iPSC suggested novel avenues for the generation of disease models and for the validation of drug targets. As an example, the work from Cowan´s group showed, for the first time, the possibility to target several genes (15) in iPSC and to study the phenotypic consequences of the gene targeting (Ding et al. 2013).

Most of the initial work with TALENs, as with ZFNs, was limited to loss of function strategy. In fact, the majority of cell lines preferentially use NHEJ to edit the genome even when an exogenous DNA donor is provided. This effect is particularly evident in non‐dividing primary cells that are the main cells targeted during clinical gene editing.

A significant amount of work was devoted to increase the efficiency of Homology Directed Repair after DSB generation and the most successful approaches required the use of single‐strand oligonucleotide (with similar homology arms and design to the Recombineering oligonucleotides) as DNA donor (Chen et al. 2011). Moreover, the use of DNA repair drug inhibitors (Maresca et al. 2013) (mainly targeting DNAPK, a key enzyme in the NHEJ pathway) was also proposed to boost HDR.

At the same time, a new logic of DNA engineering was presented by scientists at Novartis (Maresca et al. 2013) and at Sangamo (Cristea et al. 2013) that relied solely on NHEJ to promote gene knock‐in. This technology known as ObLiGaRe (when combined to ZFNs and TALENs) or as HITI (when combined to Cas9/CRISPR) (Suzuki et al. 2016) is using NHEJ to ligate compatible sticky ends or blunt ends resulting from the simultaneous cleavage of the targeting vector and the cell genome. The technology is particularly efficient in differentiated cells, CHO cells, and Zebrafish embryos. Additional knock‐in strategies were developed to use MMEJ, as an alternative pathway of DNA integration, but they are mostly efficient in transformed cells (Sakuma et al. 2016).

The development of several genome editing technologies and the limited success of functional genomics screens using RNAi set the scene for the CRISPR‐Cas9 revolution. The impact of CRISPR‐Cas9 in life science would not have been so meaningful without the improvements described above.

Historically, the CRISPR arrays were first described in 1987 (Ishino et al. 1987) and the association of CRISPR arrays to bacterial immunity and DNA targeting was documented in 2005/2006. In particular, Koonin´s group predicted that the CRISPR system could represent an RNAi‐like system and interestingly few years later it outplaced RNAi for functional genomics screening (Makarova et al. 2006). The work of Barrangou and Horvath (Barrangou et al. 2007), Sontheimer and Marraffini (Marraffini and Sontheimer 2008), and Ganeau and Moineau (Garneau et al. 2010) were all fundamental to identify CRISPR as an immune system and the CRISPR‐associated proteins (Cas), particularly Cas9, as the effector of this immune system. The subsequent identification of the noncoding RNA named TracR and the validation of the in vitro targeted cleavage of CRISPR‐Cas9 started an exciting revolution in the genome editing field. The application of CRISPR‐Cas9 system to target the mammalian genome and to induce homologous recombination or NHEJ‐mediated knock‐in proved that this system could outcompete all the previous DNA targeting technologies. The application of CRISPR‐Cas9 to engineer higher eukaryotic genomes is characterized by an extremely simplified design and a remarkable ability to induce on‐target indels. Moreover, the system is compatible with being encoded in lentiviral vectors, thus facilitating applications in functional genomics screenings (Shalem et al. 2014). The biology of the CRISPR system is discussed in detail in Chapter 3 of this book and various application throughout in many of the chapters.