Carbon Chapter 84 Cas

1 Introduction

CRISPR–Cas systems have only been discovered within the past decade during which they attracted considerable interest (van der Oost, Westra, Jackson, & Wiedenheft, 2014). The molecular understanding of some of their enzymatic components, e.g., the Cas9 protein, has been exploited to develop new tools for genome engineering and gene regulation that are easier to generate than existing technologies such as ZFNs and TALENs (Gilles & Averof, 2014). CRISPR–Cas systems are present in most archaea and in 10–40% of bacteria (Burstein et al., 2016). They are typically referred to as adaptive and heritable immune systems in the sense that microorganisms acquire resistance to extrachromosomic elements, such as viruses or plasmids (Bolotin, Quinquis, Sorokin, & Ehrlich, 2005; Mojica, Diez-Villasenor, Garcia-Martinez, & Soria, 2005; Pourcel, Salvignol, & Vergnaud, 2005). This is achieved by integrating short DNA sequences (< 40 bp) into the CRISPR loci in their genomes that act as memory of former infections (Barrangou et al., 2007; Mojica, Diez-Villasenor, Garcia-Martinez, & Almendros, 2009) (Fig. 1). This step, still poorly understood at the molecular level, is called adaptation. Each integrated sequence (also called spacer) (Grissa, Vergnaud, & Pourcel, 2007) is separated from the next spacer by a short identical repeat that often is palindromic (Lawrence & White, 2011). The name of CRISPR was first derived from the discovery of these islands, i.e., clusters of regularly interspaced repeats (Ishino, Shinagawa, Makino, Amemura, & Nakata, 1987). Following transcription of the CRISPR locus, the repeats are specifically recognized by a ribonuclease that generates small RNAs, also called crRNAs (for CRISPR–RNAs) (Charpentier, Richter, van der Oost, & White, 2015). The crRNAs are then integrated into large monomeric or multimeric protein complexes formed by the CRISPR-associated proteins (Cas proteins), which scan the cellular nucleic acids for the presence of a target sequence. When a nucleic sequence complementary to the crRNA is encountered, it will be degraded either by the ribonucleoprotein (RNP) complex itself or by recruitment of an additional factor displaying the nuclease activity. This stage is called interference and the target sequence is named the protospacer, referring to a previously encountered DNA sequence.

Fig. 1. General model for CRISPR/Cas systems. (A) Schematic view of a prokaryotic cell getting infected by a virus. In the adaptation stage, the protein complex Cas1–Cas2 (in blue) is capturing a short piece of viral DNA (in orange) which is then integrated in a CRISPR locus on its genome. The newly acquired sequence (called spacer) is integrated in-between repeat sequences (in black) just beside a leader sequence containing a promoter (in yellow). At this stage, the cell and its progeny will be immunized against the virus. The RNA CRISPR locus transcript is specifically cleaved by a ribonuclease in the repeat sequences generating small RNAs (called crRNAs), each carrying a memory of the viral sequence. A single crRNA is carried by a large protein or protein complex that will search for the complementary sequence in any cellular DNA. The recognition of a matching target (called protospacer) will lead to the degradation of the latter, either by the recruitment of an accessory nuclease or by the intrinsic activity of the RNP complex. (B) Schematic representation of the Cas genes coding for the proteins implicated in the molecular processes and of the effector complexes from three major CRISPR–Cas types. Bioinformatics studies allowed the classification of different types and subtypes of CRISPR systems. Whereas in all systems are found the adaptation proteins Cas1 and Cas2, the effector complexes involved in interference are quite different in composition. The type II is the large monomer Cas9 and the types I and III are multimeric with a common Cas7 protein forming a backbone on the crRNA. For the types I, II, and III are represented the subunits of the main studied CRISPR RNP complexes (Cascade, Csy, Cas9, CMR, and CSM) with their respective protein signatures being Cas3, Cas9, and Cas10. In contrast to type III targeting RNA with their crRNA, types I and II are targeting DNA and are generating an R-loop which will trigger the cleavage. The Cascade and Csy complexes recruit the fused helicase nuclease Cas3 whereas Cas9 induces a double-strand break by dual incision.

Bioinformatics analysis of the Cas proteins allowed the classification of the CRISPR systems into different types and subtypes. The last published classification proposed up to five types (from type I to V) (Makarova et al., 2015) among which the types I, II, and III represent the best studied systems (van der Oost et al., 2014). A common feature for all types is the presence of the proteins Cas1 and Cas2, which are involved in the capture and integration of new spacers in the adaption stage (Makarova et al., 2011; Yosef, Goren, & Qimron, 2012) as well as the presence of a crRNA-containing RNP complex for target recognition at the interference stage. By integrating crRNAs with a specifically designed sequence, these RNP complexes can be reprogrammed to recognize practically any target of choice. The different types of CRISPR–Cas systems use different RNP complexes and further distinguish themselves by the presence of a specific "signature protein" that is responsible for DNA degradation which is, respectively, Cas3, Cas9, and Cas10 for the types I, II, and III (see Fig. 1). Type I systems employ a large multisubunit RNP complex called Cascade that recognizes double-stranded DNA (dsDNA) targets. After target recognition and verification, Cascade recruits the signature protein Cas3—a fused helicase–nuclease—to degrade DNA (Sinkunas et al., 2011, 2013). In type II systems, the monomeric Cas9 protein is both the RNP for dsDNA target recognition as well as the signature nuclease for target degradation. Using its two nuclease domains, it readily generates a double-strand break on bound targets (Gasiunas, Barrangou, Horvath, & Siksnys, 2012; Jinek et al., 2012). It represents a minimal system and therefore became the preferred tool in CRISPR–Cas-based genome engineering applications (Hsu, Lander, & Zhang, 2014; Karvelis et al., 2013; Mali et al., 2013; Sander & Joung, 2014). In type III systems, the RNP complex is multimeric with a similar helicoid structure as found for Cascade (Cas7 family proteins) (Benda et al., 2014; Rouillon et al., 2013). Despite this similarity, the RNP complex is not recognizing dsDNA but complementary RNA sequences (Hale et al., 2009; Tamulaitis et al., 2014; Zhang et al., 2012). RNA recognition stimulates a nonspecific DNA cleavage activity of the Cas10 signature nuclease that is part of the RNP complex (Elmore et al., 2016; Estrella, Kuo, & Bailey, 2016; Kazlauskiene, Tamulaitis, Kostiuk, Venclovas, & Siksnys, 2016), such that DNA cleavage is achieved cotranscriptionally (Kazlauskiene et al., 2016; Samai et al., 2015).

The central and most crucial step during interference and genome editing is the recognition and the verification of the target sequence by the RNP complex. The recognition should be specific enough to avoid degradation of undesired targets (off targets). All wild-type CRISPR–Cas systems found so far are, however, somewhat promiscuous, i.e., they tolerate a number of mismatches between crRNA and target (Fineran et al., 2014). This is suggested to be beneficial during the defense against foreign DNA, since invaders (such as viruses) can less easily escape by mutations in the protospacer. Promiscuous target recognition leading to massive off targeting is however highly problematic in genome engineering applications (Wang et al., 2015; Wu et al., 2014). It is therefore crucial to understand the mechanism of target recognition and to develop experiments with which quantitative insight into this process can be obtained.

Most of the knowledge on how CRISPR–Cas systems recognize protospacer targets has been gained so far for types I and II systems. They both recognize dsDNA. In addition to a well-matching target that is complementary to the crRNA they require a short nucleotide motif upstream of the protospacer, called PAM (protospacer adjacent motif), that is recognized by the protein component of the complex (Semenova et al., 2011). PAM recognition is a prerequisite for protospacer recognition, during which the crRNA base pairs with the complementary target strand of the DNA duplex. The nontarget strand (NTS) is thereby expelled, leading to the formation of an RNA–DNA hybrid called the R-loop (Jore et al., 2011). Successful R-loop formation triggers subsequent DNA cleavage.

A broad range of techniques have been applied to decipher the molecular mechanisms of CRISPR–Cas systems. The combination of in vivo and in vitro studies, associated with structural snapshots, allowed understanding the pathways of CRISPR systems as briefly described above (Fig. 1). Among in vitro approaches, single-molecule tools have uniquely revealed the dynamics of the RNP complexes during protospacer recognition. Single-molecule fluorescence experiments, such as fluorescence resonance energy transfer (FRET) and DNA curtain assays were able to monitor protospacer binding by the types I–E surveillance complex Cascade (Blosser et al., 2015; Redding et al., 2015). Furthermore, the dynamic search of DNA targets by the type II RNP Cas9 taking place by a three-dimensional diffusion binding mechanism could be followed (Sternberg, Redding, Jinek, Greene, & Doudna, 2014). As an alternative approach, our group applied a force-based technique, specifically magnetic tweezers, to study the target recognition of CRISPR–Cas systems. Compared to the fluorescence approaches, magnetic tweezers are uniquely able to monitor in real time the formation and the extent of the actual R-loop structure. Additionally, the dependence of R-loop formation on the applied mechanical stress (torque from DNA supercoiling) can be studied (Szczelkun et al., 2014). This technique revealed unique insights into the directionality of R-loop formation, the R-loop stability, and the necessary conditions for DNA target cleavage (Rutkauskas et al., 2015). In this chapter, we provide an overview about magnetic tweezers investigations of CRISPR–RNP complexes from types I–E (Cascade complex) and type II (Cas9 complex) and explain how different aspects of the target recognition process can be dissected using this methodology.

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CRISPR-Cas Enzymes

Kwang-Hyun Park, ... Eui-Jeon Woo, in Methods in Enzymology, 2019

1 Introduction

CRISPR/Cas systems eliminate invading phages and plasmids in prokaryotes and rely on ribonucleoprotein effector complexes (Sorek, Lawrence, & Wiedenheft, 2013). CRISPR/Cas systems are divided by two classes depending on the architecture of interference effector modules and are grouped into six major types (types I–VI); at least 25 subtypes exist based on the signature cas gene and the distinct mechanism of targeting (Koonin, Makarova, & Zhang, 2017). In the class 1 system that forms multiple complexes, the Cascade complex of type I and the Csm and/or Cmr effector complexes of type III are phylogenetically related ribonucleoproteins (Makarova et al., 2015). Both complexes form a common, twisted helical structure with homologous Cas subunits (Tamulaitis, Venclovas, & Siksnys, 2017) (Fig. 1A). The class 2 system represents a single interference protein that is currently categorized into three distinct types (type II, V, and VI) (Shmakov et al., 2017). The Csm complex in type III/A consists of five subunits (Cas10 and Csm2–5) and a crRNA (Rouillon et al., 2013; Staals et al., 2014) (Fig. 1B). The crRNA is composed of eight nucleotides (nt) at its 5′ end derived from a repeat region and 25–45 nt derived from a spacer region of CRISPR loci. The 5′ end the crRNA is processed by the Cas6 protein and recognized by the Csm4 subunit in the Cas10/Csm complexes (Osawa, Inanaga, Sato, & Numata, 2015; Sokolowski, Graham, & White, 2014). The Csm complexes cleave target RNAs by catalytic activity of the backbone Csm3 subunits (Staals et al., 2014). DNA cleavage by the Csm complexes requires transcription that causes exposure of the single-strand DNA (ssDNA), then Cas10 degrades the exposed ssDNA with an RNA transcript coupled targeting mechanism (Kazlauskiene, Tamulaitis, Kostiuk, Venclovas, & Siksnys, 2016). The hyperthermophilic archaeon, Thermococcus onnurineus NA1, has six CRISPR loci in which 16 Cas proteins were identified for the two CRISPR–Cas systems: the subtype III/A and the putative IV system (Jung et al., 2016) (Fig. 1C). The type III/A system is located between CRISPR locus 3 and 4 in the chromosomal DNA with seven cas–csm genes (Ton_0892–Ton_0898) encoding cas6, cas10, csm2, csm3, csm4, csm5, and csx1 positioned in a row (Fig. 1D). The ToCsm complex was previously shown to target RNA (Fig. 1E), RNA-dependent ssDNA (Fig. 1F), and direct ssDNA (Park et al., 2017) (Fig. 1G). The method of coexpression and purification of the whole complex directly from the microorganism is widely used. The type III effector complexes are isolated by expression of all the individual subunits and a crRNA in the host cell, and the whole complex is purified using an affinity tag (Chou-Zheng & Hatoum-Aslan, 2017; Tamulaitis et al., 2014). Although these methods are effective for isolation of the functional complexes, there are some limitations to obtaining homogeneous complexes due to altered lengths of the processed crRNAs, loss of subunits during purification and nonspecific interactions of cellular proteins with the complex. Therefore, we developed an assembly method for the proteins in order to obtain homogeneous complexes in abundance. Here, we provide a protocol for purification of type III/A effector complex subunits and assembly of the ToCsm complex in vitro. This protocol is presumably applicable to other thermostable type III/A effector complex systems in microorganisms and could facilitate various functional and biochemical analyses of the effector complex.

Fig. 1. Structure of Csm complex and CRISPR/Cas system derived from T. onnurineus. (A) The electron microscopy (EM) map of the ToCsm complex (EMD-3454). The molecular model of ToCsm complex docked into EM densities with each subunit in a different color. (B) Schematic representation of the ToCsm complex bound to crRNA. The active sites of the RNase and ssDNase of the ToCsm complex are highlighted. (C) Six CRISPR loci repeats identified in the genome of T. onnurineus NA1. (D) The organization of genes of type III-A in T. onnurineus. CRISPR locus 3 and 4 are indicated (gray box). Highlighted arrows represent the csm genes encoding the ToCsm complex. The Csm complex-dependent RNase Csx1 is positioned at the end of gene array. (E) Cleavage of 40 nt target RNA by the ToCsm complex in the presence or absence of a metal cofactor, Mn2 +. Three arrows indicate the cleaved fragments on a urea polyacrylamide gel. The RNA substrate was 5′ end-labeled with 32P. (Lane C: the reaction sample without ToCsm complex). (F) Target RNA-activated ssDNA cleavage by the ToCsm complex in the presence or absence of a metal cofactor, Ni2 +. The ssDNA substrate was 5′ end-labeled with 32P. (G) Cleavage of target 100 nt ssDNA by the ToCsm complex. The ssDNA substrate was 5′ end-labeled with 32P. The arrows indicate the major fragment generated by the ToCsm complex target DNA cleavage activity.

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Genome Defense

David P. Clark, ... Michelle R. McGehee, in Molecular Biology (Third Edition), 2019

6 CRISPR Systems Are Functionally and Structurally Classified

CRISPR-Cas systems are classified based on their structural and functional characteristics. There are two classes of systems, each having different types and even subtypes. Table 20.01 summarizes the classes, types, and subtypes of the known systems. In general, class 1 systems represent a majority of the identified CRISPR systems at present and are structurally very complicated. Class 2 systems are seemingly more rare in nature (at least those that have been identified), but have more value to genetic engineering and biotechnology, and have fewer components. Table 20.02 summarizes the roles of the more common effector and ancillary proteins by type and subtype; however, numerous other proteins are involved, some of which are currently uncharacterized and certainly many are not yet discovered. Some are even referred to with different names! Research in this area is moving very quickly.

Table 20.01. General Characteristics of CRISPR Classes, Types, and Subtypes

ClassTypeSubtypesGeneral Characteristics1II-A through I-F, and I-UUses Cas3Targets double-stranded DNARequires a PAM sequenceIIIIII-A through III-DUses Cas10Introduces single-stranded breaks in both DNA and RNA targetsDoes not require a PAM sequenceIVNone characterizedLacks many conserved cas genes and often a CRISPR array2IIII-A, II-B, II-CUses Cas9 and requires both crRNA and tracrRNAProduces blunt-ended, double-stranded breaks in target DNARequires a PAM sequenceVV-A, V-B, V-CCpf1, C2c1, or C2c3 single protein effectors (sometimes called Cas12), depending on subtypeProduces staggered, double-stranded breaks in target DNARequires a PAM sequenceType V-B effector (C2c1) requires both crRNA and tracrRNAVIVI-A, VI-B, VI-CUses C2c2 (sometimes called Cas13) single protein effectorProduces single-stranded breaks in target RNAsRequires a PFS

Table 20.02. Functions of Selected Cas and Ancillary Proteins

CRISPR ProteinAssociated Type-SubtypeFunctionCas1I, II, III-A, III-B, IV, possibly VIDNA nucleaseCas2I, II, III-A, III-B, V, some VIRNA nucleaseCas3IDNA nuclease and helicaseCas4Most type I, II, VDNA nucleaseCas5I, III, IVRibonuclease that processes pre-crRNA into mature crRNACas6Most type I and type III-BRibonuclease that processes pre-crRNA into mature crRNACas7I, III, IVContains RNA recognition motif and binds crRNA, usually present in multiple copiesCas8Most type ILarge subunit of effector complex in type ICas9II onlyDNA nucleaseCas10Some type I, most type IIILarge subunit of effector complex in type IIICas12 (Cpf1)VcrRNA processing, DNA nucleaseCas13 (C2c2)VIcrRNA processing, RNA nucleaseCsm, CmrIIISingle-stranded DNA and RNA nucleasesRNase IIIIIProcesses tracrRNA and assists with crRNA maturation

CRISPR systems are divided into two classes based on the structure of the effector complex.

6.1 Class 1 Systems Use Multiprotein Effector Complexes

Class 1 systems use multiprotein crRNP effector complexes to target recognition sequences and cleave target nucleic acid. Class 1 systems include types I, III, and IV, plus several subtypes. These systems have been identified from a diverse number of bacteria and Archaea, including from the genera Archaeoglobus, Clostridium, Bacillus, Yersinia, Escherichia, Staphylococcus, Pyrococcus, Geobacter, Vibrio, and many others. The thermophiles are well represented, especially regarding type III systems. The proteins that comprise the effector complex for each type and subtype vary. The numerous ancillary proteins tend to have similar functions among the identified types and subtypes, but may not always be present. Some accessory proteins, such as reverse transcriptase, are important for the generation of new memories originating from RNA instead of DNA.

Class 1 systems include types I, III, and IV and have large multiprotein effector complexes.

The class 1 type I Cascade (CRISPR-associated complex for antiviral defense) recognizes and unwinds double-stranded DNA. Cas3 nuclease introduces single-strand breaks. Numerous other Cas proteins assist with the effector complex. Although both strands of dsDNA are recognized, only a single strand is cut (Fig. 20.10). Type III systems are mechanistically different but share similar architecture with type I Cascade, indicating a common origin. The targets for type III systems are single-stranded DNA and RNA, especially those currently involved in transcription. In fact, degradation of these targets is coupled. As dsDNA is unwound during transcription and RNA is transcribed, both the ssRNA transcript and the ssDNA coding strand are targeted (Fig. 20.11). The type IV system is new and not well characterized. However, it is known that this system lacks the usual cas genes, and even a CRISPR array in some organisms!

Figure 20.10. Class 1 Type I crRNP Effector Complex

Cascade is a large, multiprotein effector complex that recognizes one DNA strand through the complementary base pairing with guide crRNA and then Cas3 nuclease introduces a single strand break on the opposite strand.

Figure 20.11. Class 1 Type III Coupled ssRNA/ssDNA Degradation in Transcription

Invading phage nucleic acid that is actively being transcribed is targeted with Class 1 type III systems. Here, the large effector complex recognizes ssDNA and resulting ssRNA in a transcription-dependent manner. The Cas10 and Cas7-like nucleases cleave both the ssDNA and ssRNA, respectively.

Although the class 1 systems are interesting, they are not nearly as useful for genetic engineering as the more simplified class 2 systems described later.

6.2 Class 2 Systems Require Only Single Effector Proteins

Class 2 systems require only one effector protein (usually having multiple domains) to target recognition sequences and degrade nucleic acid. Class 2 systems, which include types II, V, and VI have been used extensively in genetic engineering. The more familiar CRISPR/Cas9 is a class 2, type II system. Class 2 systems have been identified from several bacterial genera, including Streptococcus, Legionella, Neisseria, Francisella, Prevotella, and others. In contrast to class 1, no class 2 systems have been identified from Archaea.

Class 2 systems include types II, V, and VI and have a single effector protein.

A typical class 2 CRISPR system locus consists of a CRISPR array plus the genes, cas1 and cas2, which encode adaptation module proteins. Similar to class 1, the genes for accessory proteins are also present within the locus. Additionally, for types II and V-B, which both use a dual RNA structure to guide the effector complex to the target, the gene for the second RNA, called tracrRNA (trans-activating CRISPR RNA), is also located in the CRISPR locus.

All type II systems require a PAM for interference, which may be located either upstream or downstream of the protospacer, depending on the subtype. The loci contain the genes for tracrRNA located immediately upstream of the array, with cas1, cas2, and cas9 genes further upstream. As discussed previously, the cas1 and cas2 genes encode adaptive module proteins. The cas9 gene encodes the DNA endonuclease effector. Cas1 and Cas2 generate the spacer and add sequence to the CRISPR array for adaptation. During interference, tracrRNA is expressed and then processed by a ribonuclease, called RNase III. A complex of tracrRNA, RNase III, and Cas9 process the pre-crRNA into crRNA. The 5′ end of tracrRNA and the 3′ end of mature crRNA have complementary sequences and ultimately base pair to form a dual tracrRNA-crRNA structure required for targeting. The effector complex containing Cas9 and dual tracrRNA-crRNA recognizes targets and introduces blunt-end breaks in double-stranded DNA (Fig. 20.12).

Figure 20.12. Class 2 Type II Mechanism

In Class 2 systems, only single effector proteins are needed. The tracrRNA, RNase III, and Cas9 nuclease generate crRNA that combines with tracrRNA to generate a dual tracrRNA-crRNA for targeting. Double-stranded DNA complementary to the guide RNA is cleaved by Cas9 nuclease, creating a blunt-end.

When CRISPR is used in genetic engineering, an artificial RNA linker is inserted between the complementary sequences of tracrRNA and mature crRNA. This generates one single-guide RNA (sgRNA). The sgRNA functions to direct Cas9 to targets for application in genetic engineering (Fig. 20.13).

Figure 20.13. Modification of Dual tracrRNA-crRNA to sgRNA

A single-guide RNA (sgRNA) was experimentally generated by introducing an artificial linker between tracrRNA and crRNA. The sgRNA works as well as the wild type dual tracrRNA-crRNA for targeting and eliminates the need for two separate RNA molecules to be delivered to the experimental system.

Some type V systems do not require tracrRNA and use a different effector protein, Cas12a, to introduce staggered-end cuts into double-stranded DNA. Cas12a has also been called Cpf1 in recent literature and has been modified for genome editing in mammalian cells. Type VI subtypes have significantly simplified loci compared to the other class 2 types. Generally, just one gene, usually just the gene for the effector protein, is upstream of the CRISPR array.

The first use of CRISPR in biotechnology occurred when CRISPR/Cas9 was used to edit genomes. Although the effector proteins from all class 2 types have been modified and used in various applications ranging from genome editing, knockouts, mutagenesis, gene regulation, RNA inactivation and editing, delivery of proteins or small molecules to DNA and RNA, and many others, the class 2 type II CRISPR/Cas9 is the most widely used. Section 20.7 surveys some applications for CRISPR technology.

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CRISPR-Cas Enzymes

Lucas Kissling, ... Martin Jinek, in Methods in Enzymology, 2019

1 Introduction

CRISPR–Cas systems mediate prokaryotic adaptive immune responses that protect against invasive nucleic acids such as viral or plasmid DNA (Mohanraju et al., 2016). These systems harbor CRISPR-associated effector nucleases that are directed by small CRISPR RNA (crRNA) guides to target and subsequently cleave complementary nucleic acid sequences (Garcia-Doval & Jinek, 2017). Due to their programmable sequence specificity, the DNA nucleases Cas9 and Cas12a have been repurposed for genetic modification in mammalian cells (Cho, Kim, Kim, & Kim, 2013; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013; Zetsche et al., 2015) and have become versatile molecular genome-editing tools (Fellmann, Gowen, Lin, Doudna, & Corn, 2017; Knott & Doudna, 2018; Savic & Schwank, 2016) for basic research and biomedical applications. When introduced into eukaryotic cells, Cas9 and Cas12a can generate programmed double-strand DNA breaks (DSBs) in the genome whose repair by endogenous DNA repair mechanisms can be exploited for generating genetic mutations or for introducing specific modifications in the vicinity of the DSB.

While both Cas9 and Cas12a originate from CRISPR–Cas defense systems, they have distinct evolutionary histories (Shmakov et al., 2017) and mechanisms of crRNA binding, DNA recognition, and DNA cleavage (Swarts & Jinek, 2018). In contrast to Cas9, Cas12a generates its own crRNA guides from long pre-crRNA transcripts (Fonfara, Richter, Bratovic, Le Rhun, & Charpentier, 2016; Swarts, van der Oost, & Jinek, 2017; Zetsche et al., 2017). This activity allows Cas12a to process several crRNAs from longer Pol II transcripts in mammalian cells, which has facilitated combinatorial mutations and simultaneous (multiplexed) gene disruptions (Vlot et al., 2018; Zhong, Wang, Li, Tran, & Farzan, 2017). Beside complementarity between the crRNA and the target sequence (named the protospacer), both Cas9 and Cas12a additionally require the presence of a protospacer adjacent motif (PAM). Whereas Streptococcus pyogenes (Sp) Cas9 recognizes a 5′-NGG-3′ PAM downstream of the protospacer sequence, Cas12a orthologs generally have a preference for a 5′-TTTV-3′ PAM (Zetsche et al., 2015). In addition, Cas12a orthologs can target DNA with "suboptimal PAMs" with reduced efficiency (Kim et al., 2017; Yamano et al., 2017). While recognition of the PAM is important for target DNA unwinding to facilitate subsequent crRNA–DNA base pairing, it constraints the range of potential genomic sites that can be targeted by each effector enzyme. Lastly, Cas9 cuts each target DNA strand with a separate nuclease domain, generally yielding blunt-ended dsDNA breaks. In contrast, Cas12a orthologs employ a single nuclease domain that catalyzes cleavage of both target DNA strands (Swarts et al., 2017) and yields a dsDNA break with 5′ overhangs (Zetsche et al., 2015). Despite these differences, both Cas9 and Cas12a can be used to efficiently generate dsDNA breaks in mammalian cells (Swarts & Jinek, 2018). The genome-editing efficiency of SpCas9 and Cas12a orthologs from Acidaminococcus sp. (AsCas12a), Lachnospiraceae bacterium (LbCas12a), and Francisella novicida (FnCas12a) varies depending on the target DNA sequence. This, combined with the different PAM requirement, makes each of these enzymes valuable additions to the genome editing toolbox.

Both Cas9 and Cas12a can be used for generating mutations in mammalian cells by exploiting the nonhomologous end joining (NHEJ) repair pathway, which can lead to the introduction of short insertions or deletions (indels) at the DSB site (Cho et al., 2013; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013; Zetsche et al., 2015). Although the NHEJ DNA repair outcome is nonrandom (van Overbeek et al., 2016), its precise nature and its effect on the function of a gene cannot be reliably predicted a priori. Therefore, large gene deletions can be advantageous for the generation loss-of-function mutations of uncharacterized genes, when suitable antibodies may not yet be available for confirming the loss of a protein product. In addition, large deletions are readily detectable, which can facilitate genotyping of homozygous mutations.

Direct delivery of in vitro reconstituted Cas9-guide RNA ribonucleoprotein (RNP) complexes has emerged as a versatile and efficient method of effecting genome editing in a number of cell types and organisms (Burger et al., 2016; DeWitt et al., 2016; Gaj et al., 2017; Kim, Kim, Cho, Kim, & Kim, 2014; Lin, Staahl, Alla, & Doudna, 2014; Paquet et al., 2016; Roth et al., 2018). The method enables editing without the introduction of foreign DNA into cells, and can lead to improved rates of on-target editing, while simultaneously reducing off-target effects. While there is a considerable number of experimental protocols available for RNP-based genome editing using Cas9 (DeWitt, Corn, & Carroll, 2017; Kwart, Paquet, Teo, & Tessier-Lavigne, 2017; Lingeman, Jeans, & Corn, 2017; Modzelewski et al., 2018), relatively little material covers the use of recombinant Cas12a RNPs for generating genetic deletions.

Here, we provide an experimental protocol for applying AsCas12a to generate gene deletions in mouse ES cells. In specific sections of this protocol, we describe how to express and purify AsCas12a, how to design crRNA guides, and how to form Cas12a–crRNA RNP complexes. Subsequently, we detail how activity of the RNP complexes can be verified in vitro and in vivo, and how RNP complexes are delivered into mouse ES cells by electroporation. Lastly, we describe how to verify the introduced mutations or deletions.

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Viral Tools for Genome Manipulations In Vivo

Boriana Marintcheva, in Harnessing the Power of Viruses, 2018

3.3.3.1 CRISPR-Based Adaptive Immunity: Components and Mechanism

The CRISPR/Cas systems (Fig. 3.13) encompasses an array of CRISPR spacers (depicted with color boxes numbered 1–6) and repeats (depicted with white boxes) flanked by genes coding for Cas proteins (depicted with blue arrows). Each spacer is a short (30–45 nt) sequence homologous to foreign DNA that has invaded the cell previously, i.e., each spacer can be viewed as a snapshot of an intruder "taken" from the surveillance camera of the bacterial immune system. The original sequence in the phage genome corresponding to the acquired spacer is designated as a protospacer. Each CRISPR array starts with a leader DNA sequence (a gray box designated with letter L) guiding the integration of new spacers.

Figure 3.13. Mechanism of CRISPR/Cas adaptive immunity.

CRISPR loci consist of short spacer sequences of phage and plasmid origin (colored numbered boxes) separated by DNA repeats (white boxes) arranged into an array beginning with a leader sequence (gray box labeled with the letter L) containing the promoter for its expression. The array is flanked by a Cas operon (blue arrows) coding for the associated protein components, which vary by number and characteristics depending on the type of the immunity system. (A) Immunization step: spacer sequences are captured as they enter the cell and integrated into the CRISPR array immediately after the leader sequence. (B) Immunity in action: the CRISPR array is transcribed in a long transcript, which is subsequently processed into individual CRISPR RNAs (crRNAs), each coding for one spacer. crRNAs form complexes with Cas RNA-guided nucleases and target them to complementary protospacer of invading bacteriophage, which is subsequently cleaved.

Reprinted from Marraffini LA, CRISPR-Cas immunity in prokaryotes. Nature 2015;526(7571):55–61, with permission from Nature publishing group.

The action of CRISPR/Cas adaptive immunity is executed in three stages: (1) spacer acquisition, (2) transcription, and (3) interference. Spacer acquisition could be considered as an equivalent of immunization in the human immune system equipping the cell to defend itself against a repeated invasion. The process of protospacer identification is best understood in CRISPR/Cas type I systems, although many details are still lacking. It is believed that fragments of phage DNA are generated by double-stranded DNA breaks randomly arising in viral replication. Protospacer sequences are presumably identified by Cas enzymes based on recognition of 2-nucleotide-long to 3-nucleotide-long motifs, known as PAMs. The spacer integration in the CRISPR array is accomplished by two coordinated cleavage/ligation reactions taking place at the 5′ end of the first repeat sequence downstream from the leader. The spacer is ligated to the cleavage-generated 5′ ends resulting in its incorporation in the CRISPR array immediately after the leader sequence in the context of two ssDNA gaps corresponding to the flanking repeats. The gaps are repaired by DNA polymerase and sealed by a ligase, which complete the spacer integration process. Cas1 and Cas2 nucleases play key role in the spacer integration process and are well conserved among most CRISPR/Cas systems described up-to-date.

The CRISPR array locus is transcribed into long precursor RNA molecule, which is processed to individual crRNA (CRISPR RNA) molecules, each containing one spacer sequence. Mature crRNAs are able to hybridize to complementary protospacers when relevant bacteriophage DNA enters the cell, thus serving as a guide for the subsequent nucleolytic cleavage and blocking the phage infection by crRNA-based interference. One can think about the transcription stage of the CRISPR/Cas immune system as a process of preparing tools capable of neutralizing future phage infections, i.e., a process conceptually resembling B lymphocyte maturation leading to the synthesis of antigen-specific neutralizing antibodies. Similarly, the interference stage conceptually resembles the pathogen elimination by neutralizing antibodies. Interference takes place by different mechanisms in different CRISPR/Cas systems. The CRISPR/Cas9 system employs a single protein, Cas9, and a transactivating RNA molecule, commonly described as tracrRNA, i.e., transactivator of crRNA. The 5′ end of the tracrRNA is complementary to the 3′ end of the crRNA, thus their base pairing results in short dsRNA segment. It is believed that tracrRNA contributes to crRNA maturation. If the sequences of the crRNA and tracrRNA are fused together the resulting RNA piece, commonly described as gRNA is completely functional. That discovery allowed for the very straightforward design of the CRISPR/Cas9 gene editing tool, which essentially requires one protein component, Cas9 (utilized in all editing reactions), one RNA component (gRNA) specific for each sequence subject to editing and one donor DNA template to be used to repair the CRISPR/Cas9 introduced dsDNA break. The simplicity of CRISPR/Cas9 system and the availability of established approaches to deliver the two needed components into target cells and multicellular organisms allowed for the fast paced advancement of CRISPR/Cas9-based applications.

Cas9 is a multidomain protein harboring PAM-recognition domain, two individual nuclease active sites along with gRNA- and target DNA-binding activities. The protein by itself is inactive until it forms a complex with the gRNA, which allows it to start scanning or "interrogating" DNA. Nuclease cleavage does not take place unless the Cas9/gRNA complex recognizes and base pairs with a protospacer and PAM sequence simultaneously. The base pairing does not need to be perfect. The PAM consensus sequence is NGG (less frequently NAG) and the spacer/protospacer base pairing tolerates mismatches. The latter is considered a feature allowing the detection of protospacers in phages that have accumulated some mutations. The self versus nonself discrimination is achieved with the help of crRNA sequences complementary to the repeats in the CRISPR arrays. If the gRNA base pairs with a protospacer and a PAM sequence, the complementary DNA is cleaved and the phage infection is prevented. If the gRNA base pairs with a spacer and a repeat (i.e., the CRISPR locus) the complementary DNA is not cleaved, thus the integrity of the CRISPR locus is preserved. Tracing the very short history of the CRISPR/Cas editing offers unprecedented opportunities to appreciate simultaneously how advance science of the 21st century is and how much intellectual effort it takes to decipher a phenomenon starting from single observations. The first publications regarding CRISPR/Cas system reported on array of repeats with unknown function in the late 1980. Over the course of the next decade, advances in sequencing resulted in accumulation of tons of microbial genomes, and eventually such repeats were identified in multiple bacterial and archeal species and their utility for strain identification recognized. In the next decade the Cas genes were discovered, and it was appreciated that the CRISPR spacers are homologous to bacteriophage DNA. Initially and it was proposed that the CRISPR system is probably analogous to the RNA interference (RNAi) mechanisms for antiviral defense in eukaryotes. As knowledge was continuing to accumulate the conceptual principles underlining, the CRISPR/Cas systems were deciphered and its function as adaptive immune system identified. Characterization of the properties of CRISPR/Cas components from various origins resulted in the development the concept of the CRISPR/Cas9 gene editing approach along with relevant tools and applications. Roughly three decades after the initial discovery of the CRISPR repeats, the scientific community is starting to discuss a CRISPR/Cas pipeline of drug discovery, which is expected to transform the capabilities of contemporary medicine (Fig. 3.14).

Figure 3.14. Envisioned pipeline of CRISPR/Cas-driven drug design.

Unmet medical needs for numerous diseases and the rapid progress of CRISPR/Cas gene editing can feed into a drug discovery and development pipeline, which leads to improved therapies. The CRISPR/Cas system allows for improved target identification and validation as well as faster generation of safety models. CRISPR–Cas can also be used to develop cell-based therapies, such as chimeric antigen receptor T cells for immunotherapy and C-C motif chemokine receptor 5 (CCR5)-knockout (KO) cells for HIV treatment. CRISPR–Cas-assisted drug discovery will yield innovative therapies and treatment paradigms for patients. SNP, single-nucleotide polymorphism.

Image and figure legend reprinted from Fellmann C, Gowen BG, Lin PC, Doudna JA, Corn JE. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat Rev Drug Discov 2017;16(2):89–100, with permission from Nature publishing group.

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CRISPR-Cas Enzymes

Joseph T. Wade, in Methods in Enzymology, 2019

Abstract

The specificity of CRISPR–Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR–Cas systems for biotechnology applications. Moreover, the specificity of CRISPR–Cas systems plays an important role in prokaryote evolution due to its role in distinguishing self from nonself. Here, I describe Library-ChIP, a high-throughput method for measuring Cas protein occupancy at many DNA sequence variants in a native prokaryotic host. Library-ChIP can be used to identify the determinants of specificity for Cas protein binding to nucleic acid targets.

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CRISPR-Cas Enzymes

Matthew A. Nethery, Rodolphe Barrangou, in Methods in Enzymology, 2019

Abstract

Pervasive application of CRISPR–Cas systems in genome editing has prompted an increase in both interest and necessity to further elucidate existing systems as well as discover putative novel systems. The ubiquity and power of current computational platforms have made in silico approaches to CRISPR–Cas identification and characterization accessible to a wider audience and increasingly amenable for processing extensive data sets. Here, we describe in silico methods for predicting and visualizing notable features of CRISPR–Cas systems, including Cas domain determination, CRISPR array visualization, and inference of the protospacer-adjacent motif. The efficiency of these tools enables rapid exploration of CRISPR–Cas diversity across prokaryotic genomes and supports scalable analysis of large genomic data sets.

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Repurpose Analysis Expanding Biomedical Benefits by Omics Data Integration

Tao Zeng, in Reference Module in Biomedical Sciences, 2019

Many Ideas Initiated by Repurpose Learned From Bacterial During the Experimental Technology Development

The technology development motivated from bacterial CRISPR-Cas9 system has shown great potential in biological, biomedical, clinical studies and applications (Dominguez et al., 2016). A diversity of native CRISPR-Cas systems indeed exist in many bacteria and most archaea (Hidalgo-Cantabrana et al., 2018). To repurpose these systems for a variety of opportunities in genome remodeling and transcriptional regulation will be beneficial to the biotechnological applications. And as a common view, the whole elements of those systems must be identified and functionally characterized in their native host ahead of any practice.

Actually, using p53 as a proof-of-principle target, the CRISPR/Cas9 genome-editing system had illustrated the capability as positive selection assays of targeted gene disruption. It possibly serves as a useful and cost-effective approach for screening situ mutagenesis in a genome-wide manner (Malina et al., 2013). In other more practices, CRISPR interference based on Cas9 has provided a simple approach for targeted gene regulation. It can selectively and efficiently interrogate, perturb, or engineer cellular systems on transcriptome level (Qi et al., 2013). Next, complementary to the conventional genetic approaches, disrupting or editing gene transcripts rather than DNA elements would provide a distinct perturb method on a genetic system, e.g. a few type VI CRISPR systems had been implemented to manipulate RNA rather than DNA by targetedly knocking down endogenous gene transcripts (Jing et al., 2018). Besides, a CRISPR-Cas9 based tool has also been implemented for epigenome editing, e.g. adjusting expression activity by changing DNA methylation's heritable state on the targeted region (Vojta et al., 2016).

Obviously, the above methods based on programmable editing from CRISPR-Cas9 are usually repurposed for usage in almost any kind of cell, and they are strongly expected to treat human disease if technical and ethical issues can be seriously addressed (Bieniasz, 2017).

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Defense Against Viruses and Other Genetic Parasites in Prokaryotes

Kira S. Makarova, ... Eugene V. Koonin, in Reference Module in Life Sciences, 2019

Genomic Organization and Evolution of Defense Systems

Defense Islands and a Tight Link between Defense and Mobilome Genes

Defense genes and typical mobilome components, such as viral, plasmid and transposon genes, show statistically significant clustering in genomic islands. Such islands can be roughly defined as genomic regions flanked by housekeeping genes and containing at least one known defense gene present. Other defense and mobilome genes and uncharacterized genes, if present in the respective genome region, are included in the island. This observation led to the prediction of many new defense genes and systems that are over-represented in such islands. Furthermore, the so-called cargo of many mobile elements includes diverse defense genes that could counter host defense, contribute to competition between mobile elements or serve as addiction modules. Defense genes are especially common in some groups of mobile elements, such as Tn7-like transposons and integrative and conjugative elements (ICEs) but, surprisingly, are relatively rare in plasmids and viruses (Fig. 3). It can be argued that all defense genes belong to regions of genome plasticity and, at some point in their evolution, could have been associated with MGE. Not surprisingly, defense genes are more prone to horizontal gene transfer (HGT) than any other functional class of microbial genes except for the mobilome itself, and multiple examples of recent acquisition of defense islands can be observed when closely related genomes are compared.

Fig. 3. Tn7 cargo genes and recruited CRISPR-Cas systems. (A). Related Tn7 loci linked to CRISPR-Cas system I-F variant. For comparison a related Tn7 locus from Vibrio parahaemolyticus S023 without I-F system is shown. (B). Related Tn7 loci linked to CRISPR-Cas system V-U5 type. For each locus, species name, genome accession number and nucleotide coordinates are indicated. The genes of a representative locus are shown by blocked arrows with the scale roughly proportional to gene length and the direction of gene transcription. Genes are color-coded according to the functional groups explained in the inset below. Gene product names are indicated if known, otherwise abbreviated protein family description is provided below or above the respective arrows. see Fig. 1 for innate immunity systems gene names. Abbreviations: dcd, dCTP deaminatase; RT, reverse transcriptase; SLATT, membrane protein frequently associated with immunity systems; TPR, tetratricopeptide repeats containing protein; CHAT, caspase family protease; HTH, helix turn helix; WYL, transcriptional regulator of respective family; Znf, zinc finger domain containing protein; DDE, transposase of respective superfamily; OLD, ATP-depended DNase of respective family; GIY-YIG, HNH, and PD-D/ExK are distinct DNases families; BECR and HEPN are PCD RNase families.

Remarkably, some of the defense systems were apparently recruited by MGEs to facilitate transposition. The most prominent example is the association of Tn7-like transposons with CRISPR-Cas systems that are competent for precrRNA processing and target binding but not for DNA cleavage that is required for interference. Such CRISPR-Cas systems are hypothesized to guide Tn7 transposition according to the spacer matches which differ from the typical attachment sites recognized by the standard Tn7 transposition machinery. Fig. 3 shows gene organization of closely related Tn7-like elements associated with either CRISPR-Cas I-F (Fig. 3(A)) or V-U5 (Fig. 3(B)) systems but carrying different defense genes as a cargo. Some MGE mobilization genes have been also recruited by some defense systems. For instance, cas1, the signature gene of CRISPR-Cas system, likely originated from a distinct group of self-replicating MGEs called casposons and RAG1, the key enzyme of V(D)J recombination of the Ig-based adaptive immunity system in vertebrates, is derived from a eukaryotic transposon of the Transib family.

Gene and Domain Shuffling and Sharing

In addition to being HGT attractors, defense systems and islands are hot spots for non-homologous recombination. This phenomenon is well-studied for the type I RM systems and is often referred to as phase variation. The XerC-like recombinase IvrR is responsible for shuffling between the target recognition domains of the hsdS specificity subunit alleles via reversible recombination process. Phase variable RM loci encompass several hsdS genes, often an ivrR gene, and either inverted or direct repeats. Another common mechanism mediating phase variation is DNA polymerase slippage that occurs in simple sequence repeat regions in the TRD region of the Res subunit of type III RM systems. Most likely, these or mechanistically similar processes are also involved in shuffling of domains, individual genes and gene modules in other defense systems. The described examples include restriction endonuclease domain shuffling in RM systems, switching between toxin and antitoxin in toxin-antitoxin systems and effector module shuffling in CRISPR-Cas systems.

Gene sharing is a less thoroughly understood phenomenon. It has been noticed that some superfamily II helicases, especially, COG1205 and COG0553, and ParB family proteins are abundant in defense islands but seemingly do not belong to a single conserved gene neighborhood. Fig. 1 shows that COG1205 helicase and the accompanying DUF1998 domain containing protein belong to core genes sets in DISARM, DPD and Druantia systems. The PLD domain containing nuclease, often encoded next to the COG1205 helicase and the DUF1998 protein, is present in both DISARM and DPD. Despite this link between COG1205 helicase and PLD nuclease in DISARM system, the former is essential for the protection from the phage infection, whereas the latter is not. The COG1205 helicase is also essential in type I Druantia system along with other four genes. Thus, this helicase appears to be an integral component of some defense systems and an ancillary gene in others. Further research is needed to determine whether these genes are essential in other systems and to decipher their specific roles.

Association of Defense Systems With Programmed Cell Death Components

Sequence and gene neighborhood analyzes have identified many domains and genes homologous to RNase toxins of TA and ABI systems in the neighborhoods of RM, CRISPR and other defense systems (Fig. 4). Many of these RNases belong to the HEPN domain superfamily. Only for a few of them, the role in defense is at least partially characterized. The best-studied example is the association of prrC anticodon nuclease gene with the type IC RM system. The PrrC proteins contains a HEPN domain and contributes to the phage T4 exclusion mechanism. Inactive PrrC is a subunit of the HsdSMR complex, but it can be allosterically activated by increased levels of dTTP or by the small anti-restriction peptide encoded by the T4 phage. Activated PrrC cleaves the anticodon of tRNALys. Phage T4 encodes an RNA repair system that consists of the Pnl and Rnl1 proteins and can reverse the damage inflicted by PrrC.

Fig. 4. HEPN domain containing PCD/dormancy RNases linked to various defense systems. The genes are shown as colored arrows. Gene names (if known) are indicated below respective arrows. Colors correspond to distinct defense system groups, which are circles by dashed lines. Multidomain proteins are colored blue, known domains are indicated below the arrows. The HEPN domain is shown by a light green shape with a red outline. Distinct families of HEPN superfamily are indicated on the right for abortive infection systems and below the respective shapes in red font for other systems according to Anantharaman et al. (see Further Reading list). For each HEPN domain containing protein locus tag of respective protein is provided. The only other PCD related RNase of RelE family is shown by purple shape with red outline. Abbreviations: HEPN, higher eukaryotes and prokaryotes nucleotide-binding domain, predicted endoribonuclease; MNT, minimal nucleotidyltransferase; HNH, PD-(D/E)xK, DEDD are nucleases from respective superfamilies. RM and CRISPR-Cas gene names and types follow current nomenclature and classification.

The second example of the defense-PCD association involves another HEPN domain containing protein that is associated with many type III CRISPR-Cas systems. In these proteins, the HEPN domain is fused to a CARF (CRISPR-associated Rossmann fold) domain and is allosterically activated through the binding of a cyclic oligoadenylate that synthesized by the Cas10 protein by the CARF domain. Once activated, HEPN cleaves RNA in a non-specific manner. This activation is reversible because cyclic oligoadenylate messengers are hydrolyzed by dedicated nucleases. Furthermore, as mentioned above, effector proteins of type VI CRISPR-Cas systems become non-specific RNases upon binding the crRNA and the cognate RNA target.

There are two hypotheses explaining the coupling of defense and PCD systems: first, dormancy induction allows the host to "buy time" for the activation of other defense systems, in particular, adaptive immunity; second, dormancy or 'altruistic' suicide can be the 'last resort' measure to prevent viral spread if other immunity mechanisms fail.

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DNA Methylation: Basic Principles

C.A. Moylan, S.K. Murphy, in Medical Epigenetics, 2016

Specific Targeting—The Future

Researchers are now working on developing ways to induce hypomethylation in a highly targeted fashion. Progress has been made in being able to modify specific epigenetic marks using several ingenious methods. The idea is to engineer proteins that have exquisite DNA-binding site specificity coupled to the active domain of an epigenetic modifying enzyme. This has been accomplished, for example, using zinc finger proteins coupled to the Herpes simplex virus VP16 activation domain to reactivate imprinted genes [140], a methyltransferase domain that targets specific sequence to add methylation [141], and using TET proteins to induce demethylation of specifically targeted genes [142]. Another unique approach has been to adapt the CRISPR/Cas system by inactivating the nuclease function of Cas9 and fusing this to the Krüppel-associated box (KRAB) repressor and targeting the hypersensitive site 2 (HS2) enhancer, leading to the addition of repressing modifications to histone proteins at the enhancer element and repressing the expression of multiple globin genes under the control of this enhancer [143]. In addition, the nuclease inactivated Cas9 fused to the catalytic core of the human P300 acetyltransferase leads to acetylation of histone proteins (an activating modification) and induced expression of target genes [144].

Altogether these exciting studies hold great promise for future development as highly specific epigenetic therapies to either repress or reactivate expression of genes whose epigenetic status has been altered and as such plays a contributing role in disease. It may be possible to define the epigenoprint of an individual's tumor, biopsy, blood, or other relevant biological specimen to determine the particular combination of epigenetic modifiers needed to treat the genes that have been disrupted to restore normal expression profiles.

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Chapter 84 - Cas

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