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The most prominent application of CRISPR systems since its discovery has been genome editing. This has stemmed from realization that Cas proteins can induce precise cuts in DNA (Marraffini and Sontheimer 2008; Garneau et al. 2010) guided by the crRNA molecule (Gasiunas et al. 2012; Jinek et al. 2012). The first protein shown to do this in a simple manner was the type II Cas9 protein of S. pyogenes. The simplicity of type II systems, where only one effector protein guided by crRNA:tracrRNA is required to introduce a guided DNA break, in contrast to type I systems which require a dozen of Cas proteins to form a functional Cascade complex, has allowed this system to be used in a plethora of eukaryotic systems to introduce precise genetic changes (Cong et al. 2013; DiCarlo et al. 2013; Ding et al. 2013; Friedland et al. 2013; Hwang et al. 2013; Jinek et al. 2013; Mali et al. 2013). The system has been further simplified by fusing crRNA to tracrRNA into a chimeric single‐guide RNA (sgRNA) (Jinek et al. 2012), thus reducing the complexity of the toolkit needed for gene editing to just two components, where the sgRNA can very easily be replaced, allowing unprecedented flexibility in gene editing. SpyCas9‐mediated gene editing has since been one of the most prolific technologies in biomedical sciences, with the application ranging from precise (epi)genome engineering, modulation of gene activity, forward genetic screens, proteomics, imaging studies, diagnostics, and therapy.

While gene editing will be addressed in detail elsewhere in this book, we would like to give the basics here. SpyCas9, guided by crRNA:tracrRNA (or more frequently sgRNA), once heterologously expressed in a cell, tissue, or organism, is guided to a specific site in the genome where upon successful recognition it will introduce a blunt‐ended DSB. The repair outcome of the DSB by the host machinery can result in mutagenic events such as insertions or deletions by the nonhomologous end joining or microhomology‐mediated end joining (optimal for generating genetic knockouts), or stimulate the introduction of desired DNA sequence via recombination with a donor sequence (desired outcome for model generation and therapy). It is therefore of utmost importance for Cas9 to specifically and efficiently introduce a break only at the desired site. Furthermore, the precision and efficiency of other gene editing methods, such as base editing (Komor et al. 2016), prime editing (Anzalone et al. 2019), or site‐specific transposition (Chen and Wang 2019), are also in part dictated by the properties of Cas protein.

As discussed previously, Cas9 binds and identifies its target by recognizing the PAM sequence and then base pairing the crRNA initially with the seed sequence, and subsequently with the remainder of the sequence, followed by activation of the nucleolytic activities of RuvC and HNH domains. A plethora of biochemical studies have unequivocally confirmed that mismatches between seed and crRNA sequence are refractory to Cas9 editing (Jinek et al. 2012). However, mismatches outside the seed sequence (i.e. 10–20 nt away from the PAM) are well tolerated and support efficient cutting (Anderson et al. 2015), allowing Cas9 to cleave at off‐target sites. Potential off‐target mutagenesis induced by Cas proteins is a serious concern in therapy and model development, in particular, as Cas9 was shown to be able to induce large deletions and chromosomal rearrangements (Kosicki et al. 2018). In order to circumvent such problems, a number of different computational and experimental methods have been developed to identify and prevent off‐target modifications (refer to Chapter 20).