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.
2.7 Novel Genome Editing Technologies
Most of the initial efforts in genome editing using CRISPR‐Cas9 were mainly reproducing targeting strategies previously demonstrated with Zinc Finger Nucleases and TALENs but at the same time obtaining much better efficiency with an easier design. Several groups showed the use of CRISPR‐Cas9 to promote integration by NHEJ and MMEJ. Joung and Liu´s groups showed that Cas9 could be coupled to FokI to increase its specificity (Guilinger et al. 2014; Tsai et al. 2014). Moreover, transient or stable epigenome editing was demonstrated, following the first demonstration of stable epigenome editing using TALENs (Amabile et al. 2016). The efficiency of the system to induce Gene Knock‐Out and Gene Knock‐In has been really impressive and novel strategies to further enrich for these editing events have been developed recently (Agudelo et al. 2017; Li et al. 2021).
The real differentiator between CRISPR‐Cas9 and the other genome editing technologies is the presence of an exposed single‐stranded DNA after Cas9/gRNA‐mediated strand invasion and target binding (Richardson et al. 2016). This presence of ssDNA is a peculiar characteristic of a D‐Loop‐forming enzyme and is at the base of one of the most relevant CRISPR technology, Base Editing (Komor et al. 2016; Gaudelli et al. 2017). The development of Base Editing, the precise DNA repair system that is using Cas9‐recruited Deaminase to target ssDNA (discussed in Chapter 14), further accelerated the applications of Genome Editing to drug discovery and to Therapeutic Genome Editing. In few years, Base Editing and derived technologies have dramatically impacted the field of functional genomics (Hess et al. 2016; Hanna and Doench 2020) and have provided a safer and more precise alternative to HDR or NHEJ‐based DNA editing In vitro (Webber et al. 2019) and In vivo (Chadwick et al. 2017; Carreras et al. 2019).
Liu´s group, the same group that developed base editing, used the peculiar D‐Loop induction of CRISPR‐Cas9 to develop an additional technology named Prime Editing (Anzalone et al. 2020), where a Reverse Transcriptase is fused to Cas9 to prime a gRNA templated editing event. Prime Editing is still in its infancy, but it will probably expand the reach of base editing.
2.8 Conclusions
Genome Editing and particularly the CRISPR‐Cas9‐based technologies had a tremendous impact in the drug discovery field. Despite all the advancements, the field of Genome Editing by CRISPR‐Cas is characterized by in fieri evolution where it is difficult to anticipate the next improvements and applications but certainly the presence of exposed ssDNAs can be used for other enzymatic functions promoting DNA integration, DNA labeling, or DNA regulation. Most of the CRISPR‐Cas technologies are exploiting a common process of “proximology,” i.e. proximal enzymology, that is having a tremendous impact not only in the genome editing field but more broadly in the Drug Discovery and Drug Development field, as exemplified by PROTACs and derivatives (Sakamoto et al. 2001).
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