Abstract

Many Streptococcus pyogenes strains harbor CRISPR-Cas loci encoding for the RNA-guided nuclease Cas9. Due to the use of this nuclease in genome editing there have been much focus in the study of CRISPR-Cas immunity in these organisms and as a result the S. pyogenes CRISPR-Cas9 system is one of the most studied and better understood. Here we review how the Cas9 nuclease mediates anti-phage immunity and how it can be repurposed for the genetic engineering of human cells and other eukaryotic organisms.

Introduction

Streptococcus pyogenes is the source of the most impactful genetic tool of the twenty first century: the RNA-guided Cas9 nuclease (Pennisi, 2013). This nuclease is widely used to introduce genetic modifications in a variety of cells and organisms, from bacteria (Jiang, Bikard, Cox, Zhang, & Marraffini, 2013) and yeast (DiCarlo, et al., 2013) to monkeys (Niu, et al., 2014) and human cell lines (Cong, et al., 2013; Mali, et al., 2013a). The high efficiency and simplicity of the Cas9 genome editing technique has accelerated the possibilities of human gene therapy.

What is the function of Cas9 in Streptococcus pyogenes? The nuclease is a central player of the adaptive immunity provided by clustered regularly interspaced short palindromic repeats (CRISPR) loci (Marraffini, 2015). These loci consist of short repetitive sequences (30-40 bp) that are intercalated by equally short sequences of viral (bacteriophage) and plasmid origin (Bolotin, Quinguis, Sorokin, & Ehrlich, 2005; Mojica, Díez-Villaseñor, García-Martínez, & Soria, 2005; Pourcel, Salvignol, & Vergnaud, 2005) called “spacers”. The presence of a spacer sequence matching the genome of a bacteriophage or conjugative plasmid prevents the infection of the host by these genetic invaders (Barrangou, et al., 2007; Marraffini & Sontheimer, 2008). Therefore, spacers provide sequence-specific immunity against bacteriophage and plasmid infection. Importantly, in a process known as adaptation, new spacers can be introduced into the CRISPR locus during infection (Barrangou, et al., 2007); in this way the CRISPR system creates a memory of the infection that is used to provide immunity in subsequent encounters with the same or a related (harboring the same spacer sequence in its genome) invader (Figure 1A).

Figure 1.

CRISPR-Cas immunity pathway. CRISPR-Cas loci contain clusters of repeats (white boxes) and spacers (colored boxes) that are flanked by CRISPR-associated (cas) genes. (A) During adaptation, new spacers derived from the invading DNA are incorporated into (more...)

How do spacer sequences provide immunity? The CRISPR locus is usually transcribed into a long precursor RNA containing repeats and spacers. This precursor is subsequently cleaved at the repeat sequences to liberate small CRISPR RNAs (crRNAs) that contain the intervening spacer sequence (Tang, et al., 2002) (Figure 1B). Cleavage is usually carried out by CRISPR-associated (Cas), repeat-specific endoribonucleases (Brouns, et al., 2008; Carte, Wang, Li, Terns, & Terns, 2008). The crRNA forms a ribonucleoprotein complex with an effector Cas nuclease, which finds its target (also known as the protospacer) through base-pairing of the spacer sequence in the crRNA and the genome of the invader and proceeds to cleave it (Gasiunas, Barrangou, Horvath, & Siksnys, 2012; Hale, et al., 2009; Jinek, et al., 2012; Jore, et al., 2011; Samai, et al., 2015; Westra, et al., 2012)(Figure 1C). The destruction of the viral or plasmid DNA stops the infection and confers immunity to the host (Garneau, et al., 2010). As it is expected from any host-pathogen interaction, this event is just the first step of the arms race between the host CRISPR-Cas systems and the extrachromosomal invaders, since bacteriophages and plasmids can evolve by generating mutations in the target site that prevent a perfect base-pairing with the spacer sequence of the crRNA (Deveau, et al., 2008). These mutations allow the invader to escape CRISPR-Cas immunity and re-establish infection. The cycle re-starts when the CRISPR-Cas system acquires a new spacer sequence that matches perfectly with the genome of the invader (Levin, Moineau, Bushman, & Barrangou, 2013).

Streptococcus pyogenes CRISPR loci

Depending on the cas gene content, CRISPR-Cas systems can be classified into six types (Makarova, et al., 2020). Bioinformatic analysis revealed that approximately one-third of S. pyogenes genomes contain two CRISPR-Cas loci that belong to the types II-A and I-C, one-third have only one CRISPR type (evenly distributed), and that one-third have no CRISPR present (Yamada, et al., 2019). Interestingly, the strains that lack the CRISPR type II-A locus have the highest number of prophages (Yamada, et al., 2019). More importantly, analysis of the CRISPR targets shows a mutually exclusive relationship between CRISPR spacer sequences and their prophage targets (Yamada, et al., 2019; Marraffini, 2010; Marraffini & Sontheimer, 2010; Nozawa, et al., 2011). For example, strain SF370 has a total of 9 spacers, of which 8 match sequences found in prophages present in other strains, and none match the endogenous Φ370.1-4 prophages. Additionally, in a small subset of S. pyogenes strains, the CRISPR array in the type II-A locus is interrupted by a prophage integration, first observed in M3 serotype MGAS315 (Nozawa, et al., 2011). Recently, it was experimentally demonstrated that a phage from this class, ΦAP1.1, integrates its genome into the CRISPR repeat using a tyrosine site-specific integrase (Varble, et al., 2021). When encountering a full type II-A CRISPR array, the phage can be inserted into any of the repeats, which neutralizes the immunity. While the spacers downstream of the prophage are not transcribed, the spacer immediately upstream of the interrupted repeat is not processed into a functional crRNA guide. Interestingly, during its lytic cycle, ΦAP1.1 expresses a small-protein CRISPR inhibitor, AcrIIA23, that disables Cas9 targeting. Presumably, this mechanism prevents the destruction of the phage during the initial stages of infection and enables successful lysogenization. Altogether, bioinformatic and experimental data suggests (i) that CRISPR immunity can prevent prophage acquisition in S. pyogenes, (ii) that phages have either acquired target mutations or evolved mechanisms to evade CRISPR immunity and lysogenize and (iii) that there is a dynamic relationship between S. pyogenes and its phages that results in the selection of strains with increased pathogenic adaptations. However, much about the relationship between the CRISPR, prophages and virulence properties of S. pyogenes strains remains to be established. It also remains unclear whether CRISPR immunity prevents conjugation of the many different ICE elements present in S. pyogenes strains (Beres & Musser, 2007). Although CRISPR-Cas systems have been shown to prevent conjugation in other organisms such as staphylococci (Marraffini & Sontheimer, 2008), no spacers have been found that match the conjugative elements of S. pyogenes.

Cas9-mediated defense

Although two CRISPR-Cas loci are present in S. pyogenes strains (Nozawa, et al., 2011), it is not known whether the type I-C system is actually functional and/or its cas genes and CRISPR sequences are expressed. In contrast, the molecular mechanism of S. pyogenes type II-A CRISPR-Cas immunity has been studied in detail. S. pyogenes SF370 contains a type II-A CRISPR-Cas system (Figure 2A) that harbors four cas genes (cas9, cas1, cas2 and csn2), six 30-nt spacers flanked at each side by 36-nt repeats and an additional gene that encodes for a trans-encoded crRNA (tracrRNA) (Deltcheva, et al., 2011). Cas9 is central for the defense provided by the type II-A CRISPR Cas system, since it has been shown to be essential for all three stages of immunity (Figure 1): adaptation (Heler, et al., 2015), crRNA biogenesis (Deltcheva, et al., 2011), and interference (Deltcheva, et al., 2011; Sapranauskas, et al., 2011).

Figure 2.

Cas9-mediated CRISPR immunity in S. pyogenes. (A) Organization of the S. pyogenes SF370 CRISPR-Cas locus. It contains four protein-coding cas genes, the tracrRNA gene and seven repeats (white boxes) intercalated with six spacer sequences (numbered, colored (more...)

During the biogenesis of the crRNA guides, the CRISPR array of repeats and spacers is transcribed into a long precursor RNA. As opposed to the type I and III crRNA biogenesis pathways, the S. pyogenes type II-A systems do not require a repeat-specific endoribonuclease to process the crRNA precursor (Brouns, et al., 2008; Carte, Wang, Li, Terns, & Terns, 2008). Instead, the precursor is then processed by the combined action of the tracrRNA, RNase III and Cas9 (Deltcheva, et al., 2011) (Figure 2B). The tracrRNA contains extensive secondary structure that is recognized and bound by Cas9. The tracrRNA also harbors a sequence complementary to the repeat sequence of the crRNA precursor. The annealing of these complementary sequences leads to the formation of a dsRNA that is cleaved at one end by RNase III. This cleavage liberates the small crRNAs from the precursor, which remain bound to Cas9 via their association with the tracrRNA. In this way, the processing of the type II-A crRNA precursor generates Cas9 molecules loaded with crRNA guides and ready to search invading DNA molecules for its targets.

Work performed in S. thermophilus determined that type II-A CRISPR-Cas immunity results in the introduction of double-strand DNA breaks (DSBs) on the genome of the invading phage or plasmid, at the target site specified by spacer sequences (Garneau, et al., 2010). In addition, genetic analysis of the S. thermophilus CRISPR system demonstrated that cas9 is the only cas gene necessary for the interference phase of CRISPR immunity (Sapranauskas, et al., 2011). This mechanism presents a potential for an “autoimmune” nucleolytic reaction against the CRISPR locus itself; i.e., how does Cas9 loaded with tracRNA and a crRNA avoid cleaving the spacer sequence from which the crRNA was transcribed? Early work in S. thermophilus revealed that the phage targets of the type II-A CRISPR-Cas system of this organism display a strong conservation of the sequence downstream of the target (Deveau, et al., 2008). The conserved nucleotides are referred to as the protospacer-adjacent motif or PAM (Mojica, Díez-Villaseñor, García-Martínez, & Almendros, 2009). The PAM is only present downstream of the target DNA, but absent from the repeat sequences; i.e., downstream of the spacer sequence in the CRISPR locus. Studies of the S. pyogenes type II-A CRISPR system confirmed the findings obtained for the S. thermophilus system: that cas9 is the only cas gene required for immunity (Heler, et al., 2015) and determined that the PAM motif sequence is NGG, although NAG can also function (Jiang, Bikard, Cox, Zhang, & Marraffini, 2013; Deltcheva, et al., 2011). Consistent with the essential role that the tracrRNA, RNase III and Cas9 play in the generation of crRNA guides and target DSBs, deletion of any of these three elements from the S. pyogenes genome enables transformation with target-containing plasmids, which are otherwise destroyed by Cas9 (Deltcheva, et al., 2011).

Biochemical and structural studies have characterized in detail the crRNA-guide nucleolytic activity of Cas9 that is essential to provide immunity to the host (Figure 2C) (Gasiunas, Barrangou, Horvath, & Siksnys, 2012; Jinek, et al., 2012). In addition to the tracrRNA, which is essential for Cas9 nuclease activity (Jinek, et al., 2012), S. pyogenes Cas9 contains six domains: an HNH nuclease domain, a RuvC nuclease domain, an α-helical lobe, an arginine-rich region, a Topo-homology domain and a PAM-recognition C-terminal domain (Jinek, et al., 2014; Nishimasu, et al., 2014). Single-molecule fluorescent experiments demonstrated that Cas9 searches for GG dinucleotide PAM sequences on the target dsDNA (Sternberg, Redding, Jinek, Greene, & Doudna, 2014). Transient binding to the PAM provides the necessary energy to unwind the dsDNA that is immediately upstream of the GG dinucleotide (Anders, Niewoehner, Duerst, & Jinek, 2014). Unwinding is followed by base-pairing of the crRNA with the seed sequence of the target. Inability to anneal leads to the quick release of Cas9, which continues sampling other DNA sequences. In contrast, if base pairing is productive, the rest of the crRNA sequence pairs with the target, forming an R-loop structure consisting of an RNA:DNA hybrid formed by the crRNA spacer sequence and its complementary DNA sequence (the target strand) and a displaced ssDNA (the PAM-containing, non-target strand) (Jiang, et al., 2016) (Figure 2C). The formation of the R-loop causes the HNH domain to undergo a conformational change that triggers the cleavage of both DNA strands, with the HNH domain cleaving the target strand and the RuvC domain cleaving the PAM-containing strand (Jinek, et al., 2012; Jiang, et al., 2016; Dagdas, Chen, Sternberg, Doudna, & Yildiz, 2017; Shibata, et al., 2017; Sternberg, LaFrance, Kaplan, & Doudna, 2015).

In addition to enabling crRNA biogenesis and cleaving the target DNA, Cas9 also functions as a transcriptional regulator of the type II-A CRISPR-cas locus of S. pyogenes (Workman, et al., 2021). This activity is mediated by the “long form” of tracrRNA, which is transcribed from a second promoter upstream of the promoter that generates the regular tracrRNA (Deltcheva, et al., 2011). The extra sequence enables the long tracrRNA to fold into a tertiary structure that exposes a sequence complementary to a region of the cas operon promoter. Using this atypical guide RNA, Cas9 binds to its own promoter to repress its transcription and that of the downstream cas genes. The high cas gene transcription that results from mutations introduced to interfere with this repression lead to exacerbated levels of spacer acquisition, including from the S. pyogenes genome itself. Since self-targeting spacers are known to be highly genotoxic (Jiang, Bikard, Cox, Zhang, & Marraffini, 2013), it is believed that repression has evolved to limit CRISPR “autoimmunity”.

Generation of a memory of infection

In contrast to the crRNA biogenesis and Cas9 nuclease activity, the acquisition of new spacers upon phage infection by the type II-A CRISPR-Cas system of S. pyogenes is less understood. The original observation of spacer acquisition was done in Streptococcus thermophilus, where lytic phages were added to liquid cultures and phage-resistant bacteria were isolated (Barrangou, et al., 2007). The study showed that many of the bacteriophage-insensitive mutants (BIMs) expanded the CRISPR array by the incorporation of a new spacer with a sequence matching the genome of the infecting phage. Recently, similar findings have been demonstrated in S. pyogenes (Beerens, et al., 2021); however, studies aimed at defining the steps of spacer acquisition are difficult to carry out due to the difficulties of genetic manipulation of this organism. To overcome this, a number of studies have transplanted the type II-A CRISPR-Cas system from S. pyogenes SF370 to Staphylococcus aureus (Heler, et al., 2015). Using staphylococcal lytic phages, multiple aspects of acquisition of new spacers in this CRISPR-Cas system have been elucidated.

In one instance, this arrangement was used to investigate the mechanism behind the sampling of spacer sequences with functional flanking PAM sequences (Heler, et al., 2015). Two scenarios are possible: (i) only spacers with the required NGG flanking nucleotide sequence are acquired, (ii) any phage sequence can become a new spacer but only those flanked by NGG PAMs can provide immunity and therefore are selected during phage infection. Genetic and biochemical analysis demonstrated the first scenario, with the PAM-binding domain of Cas9 being required during spacer acquisition to determine phage sequences that are flanked by a correct PAM (Heler, et al., 2015). This is a simple mechanism in which the same enzyme that is responsible for the recognition of the PAM during the implementation of immunity, Cas9, is also used to ensure that new spacers are flanked by such sequences.

The same heterologous system was used to determine, through single gene deletions, that all four cas genes (cas9, cas1, cas2, and csn2) of the S. pyogenes system are necessary for spacer acquisition (Heler, et al., 2015). While Cas9 is required for the selection of spacers with the correct flanking PAM, Cas1, Cas2, and Csn2 are suspected to be required for the next step of the CRISPR adaptation process: the integration of this sequence into the CRISPR array as a new spacer. While this has not been investigated in S. pyogenes, experiments with Escherichia coli demonstrated that Cas1 and Cas2 form a complex that is necessary and sufficient to integrate new spacers (Arslan, Hermanns, Wurm, Wagner, & Pul, 2014; Nuñez, et al., 2014; Nuñez, Lee, Engelman, & Doudna, 2015). This suggests that Cas1 and Cas2 also perform spacer integration in S. pyogenes, possibly through a more elaborate mechanism than that of the type I-E. coli CRISPR-Cas system since Csn2 and Cas9 (which are present in the type II-A CRISPR-Cas system from S. pyogenes but not in the type I system of E. coli) also form a complex with Cas1 and Cas2 (Heler, et al., 2015; Jakhanwal, et al., 2021; Xiao, Ng, Nam, & Ke, 2017). As explained above, the function of Cas9 in this complex is presumably to sample PAM sequences; the function of Csn2 is unknown (Arslan, et al., 2013).

Expression of the S. pyogenes system in a S. aureus host has brought understanding of which DNA molecules are better substrates for spacer acquisition. In one study, infection with a staphylococcal cos-phage led to the discovery that spacers are acquired immediately after genome injection, starting at the cos site; i.e., from the free viral DNA end that is inserted first into the host (Modell, Jiang, & Marraffini, 2017). This “first-end” mechanism ensures that the majority of the cells in the adapted population also target the phage immediately after genome injection, which leads to a more efficient CRISPR immune response (Modell, Jiang, & Marraffini, 2017). In a second study, it was found that the free DNA ends that are generated after Cas9 cleavage of the phage genome are also preferred spacer substrates (Nussenzweig, McGinn, & Marraffini, 2019). This mechanism equips host cells that are already immune with additional spacers and prepares them for the rise of phage escapers; i.e., phages harboring mutations that prevent cleavage at the original Cas9 target site.

Finally, spacers are typically integrated into the first position (5’ end) of the CRISPR locus, closest to the promoter of the CRISPR array. Using the heterologous system described above, it was demonstrated that this polarity is mediated by a conserved sequence immediately upstream of the array (McGinn & Marraffini, 2016), named the “leader-anchoring sequence” (LAS). Mutant cells with mutations in the LAS incorporate new spacers in the middle, not the 5’ end, of the CRISPR array. Using this mutant, it was found that the integration of new spacers in the first position of the CRISPR array mediates a stronger immune response than when spacers are integrated in the middle, which is presumably due to the higher levels of transcription of spacers at the 5’ end of the array.

Cas9-based genetic applications

Since the elucidation of the crRNA-guided DNA targeting mechanism (Garneau, et al., 2010; Marraffini, 2010), Cas nucleases have been proposed as useful biotechnological tools that require sequence-specific cleavage of DNA (Marraffini & Sontheimer, 2008; Sontheimer & Marraffini, 2010). One such application is the genetic engineering of eukaryotic cells. Early work with the yeast Saccharomyces cerevisiae demonstrated that the introduction of DSBs results in the generation of indels at the cleavage site after repair of the break via non-homologous end joining (NHEJ) (Plessis, Perrin, Haber, & Dujon, 1992; Rudin, Sugarman, & Haber, 1989). In addition, if the appropriate DNA template is provided after chromosomal cleavage, it could be used by the homology-directed repair (HDR) mechanism. In this way specific point mutations contained by the repair template can be introduced into the genome (Choulika, Perrin, Dujon, & Nicolas, 1995; Rouet, Smih, & Jasin, 1994). Therefore, a central aspect of this technique is the generation of a DSB at the desired sequence. However, the tools for this have been difficult to engineer. Sequence specific nucleases such as zinc-finger (Bibikova, et al., 2001) and TALE nucleases (Christian, et al., 2010) were developed for this purpose, but programing them to achieve sequence-specific cleavage is difficult. In contrast, S. pyogenes Cas9 provides a simple and robust tool for the generation of precise DSBs, since the specificity can be easily programmed with the crRNA guide (Figure 3). To make the system more amenable for genome editing, the tracrRNA and crRNA can be fused into a single-guide RNA (sgRNA) (Jinek, et al., 2012), thus reducing the number of components that need to be transferred from S. pyogenes to the target organism from three (tracrRNA, crRNA and Cas9) to two (sgRNA and Cas9).

Figure 3.

Cas9-mediated genome editing. Engineered Cas9 containing nuclear localization signals is expressed in human cells along with a single-guide RNA (sgRNA) to introduce a sequence-specific double strand DNA break (DSB) in the gene to be mutated. In the absence (more...)

Genome editing of human cells mediated by S. pyogenes Cas9 was first achieved by transfecting a vector harboring the sgRNA and the cas9 genes into HK293 cells to generate indels in the EMX1 chromosomal gene (Cong, et al., 2013) or in a gfp reporter gene (Mali, et al., 2013b) through the NHEJ pathway. In addition, it was shown that using an appropriate template for HDR specific point mutations can be introduced into the EMX1 gene (Cong, et al., 2013). For this, the expression of cas9 was driven by the elongation factor 1α (EF1 α) promoter, its codons were changed for optimal translation in human cells, and nuclear localization signals were added to direct the nuclease to the cell nucleus. Gene knock-out through NHEJ-mediated indel generation is highly efficient and therefore can also be performed using libraries of sgRNAs that target the whole human genome to facilitate forward genetic experiments (Shalem, et al., 2014; Wang, Wei, Sabatini, & Lander, 2014). In this approach, a library of lentiviral vectors carrying cas9 and multiple sgRNAs is used to infect cells. Upon integration of the lentiviral vector into the human genome Cas9 introduces a sgRNA-specific indel, with each cell expressing a different sgRNA and therefore having a different gene knock-out. This genetically heterogeneous cell population can be subject to different selection pressures that favor or disfavor different genotypes. Next generation sequencing of the lentiviral sgRNA locus of the cells under selection allows the identification of the gene knock-outs that are enriched or depleted, thus assigning specific genes to be responsible for particular phenotypes. Other versions of the Cas9 technology were developed by direct injection of cas9 mRNA and sgRNA molecules (Wang, et al., 2013) or of the Cas9 nuclease loaded with a sgRNA (Sung, et al., 2014). These techniques have been implemented to mutate multiple organisms, including mice (Wang, et al., 2013), flies (Gratz, et al., 2013), worms (Friedland, et al., 2013), livestock (Tan, et al., 2013), monkeys (Niu, et al., 2014), and many more. Moreover, there is the potential to utilize the technology to mutate the human germline, something that can have important ethical consequences and requires serious study (Baltimore, et al., 2015).

The simplicity of the Cas9 DNA recognition mechanism has been exploited successfully to develop other applications besides genome editing. The cleavage of bacterial chromosomal sequences by S. pyogenes Cas9 is lethal (Bikard, Hatoum-Aslan, Mucida, & Marraffini, 2012), presumably because most bacteria lack NHEJ repair (Shuman & Glickman, 2007) and the nuclease repeatedly cleaves the target every time it is repaired by HDR. This lethality has been exploited to select for bacteria carrying mutations that prevent Cas9 cleavage (such as in the PAM or seed sequences) and thus enhance bacterial mutagenesis protocols (Jiang, Bikard, Cox, Zhang, & Marraffini, 2013), as well as to develop sequence-specific antimicrobials (Bikard, et al., 2014; Citorik, Mimee, & Lu, 2014). In addition, S. pyogenes Cas9 can be converted into an RNA-guided dsDNA binding protein if key residues are mutated in each of the nucleolytic active sites (D10A in the RuvC domain; H840A in the HNH domain). This “dead” protein, or dCas9, can be fused to different functional domains to and bring them to specific sequences of the human genome. For example, the binding to promoter sequences of dCas9 fused to transcription activators or repressors can be used to modulate gene expression in human cells (Qi, et al., 2013). Similarly, the binding of fusion proteins consisting of dCas9 and chromatin modification enzymes can lead to the modification of nucleosomal histones for the silencing or activation of particular chromosomal regions (Hilton, et al., 2015; Kearns, et al., 2015). In addition, dCas9-Gfp fusions can be used to fluorescently mark different loci (Chen, et al., 2013).

Conclusions

Although the diseases caused by S. pyogenes produce a great deal of human suffering, this organism is also the source of Cas9, a nuclease that holds an enormous promise for human genome editing and gene therapy. Thus, an organism responsible for some of the most prevalent infectious diseases in the world ironically could harbor the cure for a number of genetic diseases. This highlights the importance of the research performed with S. pyogenes and other bacterial pathogens, which not only provides new therapies to combat disease, but also new genetic tools that can revolutionize medicine. Because of the revolution in human genetics caused by Cas9, much of the recent research on the CRISPR-Cas system of S. pyogenes has been centered at the biochemistry and structure of this nuclease. Future work will address the function of the CRISPR-Cas locus in the ecology, evolution and pathogenesis of S. pyogenes.

Acknowledgements

Dr. Marraffini is supported by a NIH Director’s New Innovator Award (1DP2AI104556-01). Dr. Marraffini is an Investigator of the Howard Hughes Medical Institute.

We thank the earlier authors of this chapter for their contributions to the current edition.

Abbreviations

BIMs, bacteriophage-insensitive mutants

Cas, CRISPR-associated protein

CRISPR, clustered regularly interspaced short palindromic repeats

crRNA, CRISPR RNA

DSBs, double-strand DNA breaks

HDR, homology directed repair

ICE, integrated chromosomal element

NGG, PAM sequence for S. pyogenes Cas9

NHEJ, non-homologous end joining

PAM, protospacer-adjacent motif

sgRNA, single-guide RNA

TALE, transcription activator-like effector

tracrRNA, trans-encoded crRNA

ZFN, Zinc-finger nuclease

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