Crispr Cas9

12 Key excerpts on "Crispr Cas9"

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  OMICs-based Techniques for Global Food Security

  • Sajid Fiaz, Channapatna S. Prakash(Authors)
  • 2024(Publication Date)
  • Wiley

    (Publisher)

  Genome editing offers a gateway to address previ- ously unattainable aspirations, such as correcting disease-causing mutations and enhanc- ing desirable traits. The confluence of genomics and genome editing holds the potential to redefine the boundaries of scientific achievement, propelling us into an era of customized genetics and tailored interventions (Carr and Church, 2009). Central to the current revolution in genome editing is the revolutionary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system, a microbial immune system adapted for genome engineering (Araldi et al., 2020). Originally discovered as an intriguing element in the bacterial immune system, CRISPR-Cas has rapidly emerged as a formidable tool in the hands of scientists, promising an array of groundbreaking applications. The CRISPR-Cas system’s centerpiece is the Cas9 protein, which functions as molecular scissors capable of cleaving DNA at specific target sequences. This precise targeting is guided by a single-guide RNA (sgRNA), which can be customized to direct Cas9 to any desired genetic location. The beauty of CRISPR technology lies in its remarkable versatility and accessibility. Unlike earlier genome editing techniques, the OMICs-based Techniques for Global Food Security, First Edition. Edited by Sajid Fiaz and Channapatna S. Prakash. © 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd. 160 8 CRISPR System Discovery, History, and Future Perspective simplicity of designing and implementing sgRNAs enables researchers to target specific genes with unprecedented ease and precision. The CRISPR revolution has transcended traditional boundaries, infusing diverse scientific realms. From fundamental biological research to therapeutic interventions, from agricultural advancements to unraveling the secrets of evolution, CRISPR has emerged as the harbinger of transformative change.

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  CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases

  • Luis María Vaschetto(Author)
  • 2022(Publication Date)
  • CRC Press

    (Publisher)

  CHAPTER 1 The CRISPR/Cas9 Genome-editing System_ Principles and Applications

  Cecilia Pop-Bica1 , Andreea Nutu1 , Roxana Cojocneanu1 , Sergiu Chira1 and Ioana Berindan-Neagoe1 ,2 *

  1 Research Center for Functional Genomics, Biomedicine and Translational Medicine, “Iuliu-Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania

  2 Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof. Dr. Ion Chiricuţă”, Cluj-Napoca, Romania

  * Corresponding author: [email protected]

  Introduction

  CRISPR/Cas9 represents an adjustable and widespread genome-editing tool adapted from the bacterial immune system, which is presently used in basic and applied biomedical research. Genetic alterations are frequently the cause of genetic diseases. Even though recent technological advances eased the discovery of disease-associated gene alterations, general therapeutic options are usually designed to treat the symptoms, not to restore the altered genetic sequences responsible for the disease phenotype. In this context, gene therapies appeared to support the concept of restoring the genetic mutations in order to prevent or treat the disorders caused by these alterations (Mirgayazova et al. 2020 ). In what concerns the CRISPR/Cas system (clustered regularly interspaced short palindromic repeats-CRISPR associated), the fundamental principle includes genome editing and the regulation of physiological phenomena of various organisms and cells. This system was first discovered in bacteria, where it functions as an adaptive “immune” strategy used to destroy foreign genetic material or develop resistance to phage infections (Wright et al. 2016 , Yadav et al. 2021 ). The CRISPR/Cas systems is characterized by a sequence of about 20-50 bp organized as direct repeats, isolated by spacers of comparable length, and tailed by an AT-rich “leader” region (Jansen et al. 2002 , Kunin et al. 2007 ). This system has two main components – a guide RNA (gRNA), comprised of a CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Figure 1 and Figure 2 ), which acts as a guide RNA for the complementary DNA sequence, and the Cas proteins (encoded by cas genes), which are involved in the degradation of the target DNA/RNA sequence (Strich and Chertow 2019 ). The principle of the complementary binding of the crRNA prompted the researchers to use this system to design sequences that would bind to and cut the sequences of interest in different pathologies (Jinek et al. 2012 ). Furthermore, this technology allowed researchers to identify the function of a gene through the introduction of genetic alterations (Shalem et al. 2015 ). The reduced time-frame needed to perform CRISPR/Cas9 gene editing, together with the reported efficiency of this system in terms of reduced off-target effects, makes this a superior technique compared to others based on ZFNs (Zinc Finger Nucleases) and TALENs (Transcriptor Activator-Like Effector Nucleases), in which the efficiency is exclusively based on the nucleases affinity and specificity (Qu et al. 2013

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  Transgenic Crops

  Emerging Trends and Future Perspectives

  • Muhammad Sarwar Khan, Kauser Abdulla Malik, Muhammad Sarwar Khan, Kauser Abdulla Malik(Authors)
  • 2019(Publication Date)
  • IntechOpen

    (Publisher)

  Thus, CRISPR/Cas9 technology has changed the way we see the future of agriculture. On the other hand, the implementation and easy accessibility to the CRISPR/Cas9 technology has allowed the generation of diverse molecular methodologies that constitute significant advances in the genome edition and its subsequent exploitation for agrarian and health purposes. Therefore, this technique is considered a revo-lutionary tool. The major challenges for CRISPR/Cas9 technology will focus on two underlying aspects. First, the corresponding ethical or bioethical discussion, in order to demarcate what should or should not be done with this tool considering the risks that we could face by using promising technology, an even more when this is accessible and cheap. On the other hand, the legal consequences in terms of intellectual property that today literally generate wars between law firms and universities for the patents generated by thousands of investigations must be con-sidered. Although many scientists consider CRISPR/Cas9 system as a “ Holy Grail of genetic engineering, ” we must not lose sight of the objectivity and rationality when interpreting the consequences of its use. Additionally, demanding compliance with all the necessary safety steps before this technology becomes a trivial routine, especially if this tool is used for genome editing, and the genetic improvement of living beings must be imperative. The features of the CRISPR/Cas9 system have allowed opening the possibility of using it to perform gene and cell therapy, in addition to its application in plant genetic improvement. In general, this technology has been used as a tool to perform point mutations, homologous recombination by HDR, and silencing and activation or repression of gene transcription.

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  DNA-targeting Molecules as Therapeutic Agents

  • Michael J Waring(Author)
  • 2018(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  Chapter 15 CRISPR Highlights and Transition of Cas9 into a Genome Editing Tool Thomas Bentin Department for Cellular and Molecular Medicinel, University of Copenhagen, Blegdamsvej 3c2200 Copenhagen N, Denmark

  Email: [email protected]

  15.1 Introduction

  In a book devoted to DNA recognition, it is impossible not to mention clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR associated genes (cas ). CRISPR–cas systems have enabled routine human cell genome editing in research laboratories, rapidly advancing our understanding of the human genome and raising hopes for the development of tools to address outstanding clinical challenges. The history of the discovery of CRISPR–cas ,

  1 –3

  its transition into a genome editing tool4 and its possible transfer into clinical use5 have been recently reviewed. Here, are highlighted key experiments that underpinned the discovery and transition of CRISPR–cas into a human genome editing tool.

  CRISPR–cas systems provide immunity in archaea and bacteria towards bacteriophages (phages, viruses that infect bacteria) and invasive plasmids. Immunity occurs in three steps: (i) adaptation, during which foreign DNA segments are incorporated into the CRISPR locus providing genetic memory, (ii) expression, where short CRISPR RNAs (crRNAs) are generated and (iii) interference, where the invading genome is targeted via crRNA-directed cleavage.

  The majority of sequenced archaea genomes and half of the bacteria genomes include CRISPR–cas systems. Current classification divides CRISPR–cas systems into classes, types and subtypes according to cas gene composition and architecture.6 Class 1 CRISPR–cas systems, encompassing types I, III and IV, involve multiprotein crRNA–effector complexes. Class 2 CRISPR–cas

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  Genome Engineering via CRISPR-Cas9 System

  • Vijai Singh, Pawan K. Dhar(Authors)
  • 2020(Publication Date)
  • Academic Press

    (Publisher)

  2.5. Application of CRISPR/Cas9 systems

  CRISPR/Cas9 technology initiated novel epoch of genetic engineering, which open an unprecedented opportunities for precise genome editing. Since it was first harnessed, Cas9 technology has been successfully used in medicine, agriculture and (synthetic) biotechnology for diverse applications. CRISPR/Cas9 technology has surpassed other previous prevalent genome editing method such as ZFN (Carroll, 2011 ) and TALEN (Chaudhary et al., 2016 ; Joung and Sander, 2013 ), due to its simplicity, inexpensive, specificity, easy to design and amenability to multiplexing (Wright et al., 2016 ; Doudna and Charpentier, 2014 ). Further, Cas9 was repurposed by induced a single mutations in its catalytic domain RuvC (D10A) and HNH (H840A) generate catalytically inactive also called dead Cas9 (dCas9). Dead Cas9 has no nuclease activity but it can bind to DNA. dCas9 can be coupled to transcriptional activation domain (VP16/VP64) or repression domain (KRAB/SID) to mediated transcription regulation (CRISPRa/CRISPRi) (Qi et al., 2013 ; Gilbert et al., 2013 ). CRISPRi is a more specific technology for gene silencing as compared to RNAi. dCas9 has also been used to modulate transcription state of specific genomic loci, assessing epigenetic state (Bikard et al., 2013 ; Hilton et al., 2015 ) Also, by fusing dCas9 with fluorescent marker like GFP (green fluorescent protein) has been used for labeling specific chromosomal loci, providing another method for live cell tracking (Chen et al., 2013 ). Another modification of Cas9 called base editor, a system in which dCas9 fused with cytidine deaminase (convert cytosine to uracil), which modify specific base in precise way (Kim et al., 2017a ). The base edirors has been applied in a various organisms including maize, wheat, rice, watermelon, zebrafish, mouse and Bombyx mori (Zong et al., 2017 ; Shimatani et al., 2017 ; Hua et al., 2018 ; Tian et al., 2018 ; Zhang et al., 2017 ; Kim et al., 2017b ; Li et al., 2018

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  Modern Prometheus

  Editing the Human Genome with Crispr-Cas9

  • James Kozubek(Author)
  • 2016(Publication Date)
  • Cambridge University Press

    (Publisher)

  . . Crispr refers to the repetitive nature of the repeats in the Crispr arrays that encode crRNAs, and the term does not relate directly to genome engineering. Nonetheless we prefer to use ‘Crispr-Cas9’ in a way that is less restrictive than other nomenclatures that have been used in the field.” 64 Doudna’s patents were filed under the regents of the University of California, the University of Vienna, and Emmanuelle Charpentier as a provisional application on May 25, 2012, shortly before the Jinek paper was published online in Science on June 28, 2012, describing the bio- chemistry and showing the guide RNA technology could be deployed to cut DNA in a test tube, suggesting it could be a genome editing tool (see Figure 1). In that moment, it was impossible to ignore that Crispr-Cas9 was a paradigm new technology. Amy Maxmen declared “it was elegant and cheap. A grad student could do it.” 65,66 In February 2011, Feng Zhang “heard a talk about Crispr from Michael Gilmore, a Harvard microbiologist, and was instantly captiv- ated. He flew the next day to a scientific meeting in Miami, but remained holed up in his hotel room digesting the entire Crispr litera- ture. When he returned, he set out to create a version of S. thermophilus Cas9 for use in human cells.” 67 Zhang started reaching out to experts in the field for support and collaboration, including Luciano Marraffini, crispr, cas and capitalists 21 who confirmed to me in an email that Zhang “was inspired and sought my help to carry on these experiments.” Over the next year, Zhang was able to select various versions of tracrRNA and Cas9 endonucleases from the microbes S. pyogenes and S. thermophilus, which worked the best in mammalian cells, and improved upon their fusion into a single guide RNA by restoring a small, but critical, bit of hairpin code in the tracrRNA that enabled it to function much more efficiently.

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  Food Security and Plant Disease Management

  • Ajay Kumar, Samir Droby(Authors)
  • 2020(Publication Date)
  • Woodhead Publishing

    (Publisher)

  In prokaryotes, the CRISPR is used to precisely recognize and destroy identical viral DNA fragments from viruses that has previously infected the prokaryote, using Cas protein Viz., Cas9. Cas9 uses RNA-guided mechanism that can be useful for developing successful technologies for genome editing and gene expression studies (Pennisi, 2013XXX). CRISPR/Cas in prokaryotes has contributed to a revolution in precise genome editing with an affordable, fast, effective, and rapidly flourishing technique of high precision and accuracy (Islam, 2019). Originally, CRISPR/Cas system interference was expected to be RNA-mediated and protein-dependent while later it was known that it causes sequence-specific DNA cleavage as initial mode of action (Makarova et al., 2006XXX ; Garneau et al., 2010XXX). This chapter offers a detailed overview of the theory and updates the application of the CRISPR/Cas for plant genetic modification to develop climate smart food crop varieties against various stresses and for nutritional improvement to ensure global food and nutritional stability. 9.2 Insights of CRISPR/Cas system invention The CRISPR/Cas research got going in 1987 with the discovery of repetitive DNA segment in Escherichia coli (Ishino et al., 1987XXX) and latter in other groups of bacteria and archaea (Mojica and Rodriguez-Valera, 2016XXX). CRISPR/Cas provides protection to the bacteria and archaea against the invading viruses (Gasiunas et al., 2012). CRISPR/Cas system consist of two components, first one is 29 bp short identical palindromic DNA repeats, which are regularly interspaced and second one is protospacer, which is nothing but a 32 bp hypervariable spacer DNA that is unique and matches up exactly with the bacteria infecting viruses (Hsu et al., 2014)

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  Engineering Disease Resistance in Plants using CRISPR-Cas

  • Zulqurnain Khan(Author)
  • 2023(Publication Date)
  • CRC Press

    (Publisher)

  Xie & Yang, 2013 ). Using the nonhomologous end-joining [NHEJ] or the homology direct repair [HDR] method, the system may be used to repair damaged DNA. When infected with viruses, bacteria incorporate smaller segments of the viral DNA into their own genome in a specific pattern typically known as CRISPR arrays, which is implicated in the recognition and elimination of viruses following subsequent infection. The general mechanism behind the capability of Crispr Cas9 to target viral DNA involves the generation of RNA segments from CRISPR arrays which ultimately identify and bind with specific regions of viruses’ DNA. The bacteria then utilizes Cas9 protein or similar enzyme to introduce a cut into the DNA with the consequential disabling of viruses. These processes have recently been improved to function as a more sophisticated tool for molecular biology and genetic engineering.

  History of CRISPR-Cas

  In 1987, microbiologists found several repetitive sequences in the E. coli genome that were linked with distinctive sequences known as clustered regularly interspaced short palindromic repeats (CRISPR). They discovered a novel genetic makeup consisting of alternating repeat and non-repeat nucleotide sequences similar to the sequence of that gene, although its molecular significance was not yet known. The goal of these amazing mechanisms could not be fully understood for 20 years (

  Ishino et al. 2018

  ). Experimental data proved CRISPR to be a key component of the bacterial defense mechanism against bacteriophage infection. Two distinct CRISPR loci were found in separate strains of Streptococcus thermophilus by researchers (

  Morange et al. 2015

  ). The CRISPR system spacer sequences were sequenced, and it was discovered that they matched a number of bacteriophage or plasmid sequences exactly (

  Xue et al. 2015

  ). This gave rise to the hypothesis that CRISPR-Cas was a bacterial defense system against encroaching species (Mojica & Rodriguez‐Valera, 2016

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  Transgenic Insects

  Techniques and Applications

  • Mark Quentin Benedict, Maxwell J Scott, Mark Quentin Benedict, Maxwell J Scott(Authors)
  • 2022(Publication Date)
  • CAB International

    (Publisher)

  Fig. 7.1C ).

  Fig. 7.1. CRISPR-Cas9 is a bacterial immune defence system and has been co-opted as a genome engineering tool. (A) Genomic CRISPR locus from S. pyogenes containing the operon of cas genes and the CRISPR array of identical sequence repeats (blue boxes) alternated with short invader DNA sequences (coloured diamonds). Upstream of the Cas operon, we find the tracrRNA locus. (B) The bacterial antiviral defence involves association of the Cas9 protein with precursor tracrRNA:crRNA duplexes followed by co-processing of the RNA by Ribonuclease III. (C) Mature Cas9-tracrRNA:crRNA complexes bind target viral DNA at a site complementary to the crRNA and adjacent to a PAM sequence, where Cas9 creates a dsDNA break. (D) Cas9/gRNA cleavage of genomic DNA is repaired by the cellular DNA repair mechanisms: either non-homologous end joining (NHEJ), an error-prone process that introduces mutations or deletions, or homology-directed repair (HDR), a process that uses a DNA molecule as template for repair. As a genome editing tool, we take advantage of these two DNA repair mechanisms to create mutations, deletions and insertions in a chosen cell or organism. Diagrams adapted from Doudna and Charpentier (2014) .

  The evolution of the invader–host relationship between bacteria and their infectious viruses has given rise to three CRISPR/Cas systems which differ in the molecular mechanisms of DNA recognition and cleavage (

  Makarova et al., 2011

  ;

  van der Oost et al., 2014

  ). In type I and type II systems, the specific recognition of foreign DNA from bacterial genomic sequences requires the presence of a protospacer adjacent motif (PAM) in the invader’s genome, which is a short sequence adjacent to the target sequence to which the Cas enzyme binds (

  Shah et al., 2013

  ). A special feature of type II systems is that they use two RNA molecules for recognition and cleavage of invader DNA. Indeed, this system requires a trans-activating crRNA (tracrRNA), a small RNA molecule that is trans encoded upstream of the type II CRISPR locus and that plays a role in maturation of the crRNA. The tracrRNA is partially complementary to and binds the pre-crRNA forming an RNA duplex, which is cleaved by RNAse III to form a mature duplex that acts as a guide for the Cas protein to cleave a specific sequence in the invading nucleic acid (Fig. 7.1B). The type I and type III CRISPR/Cas systems use a large complex of Cas proteins for recognition and cleavage of foreign DNA, whereas the type II system requires only a single Cas enzyme for specific genomic cleavage. Furthermore, the best-studied Cas effector protein is Cas9 from Streptococcus pyogenes, a large enzyme with two nuclease domains that introduces dsDNA breaks in invading DNA at a site within a specific 20-nucleotide (nt) sequence complementary to the crRNA and adjacent to the PAM (

  Gasiunas et al., 2012

  ;

  Jinek et al.

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  In Silico Dreams

  How Artificial Intelligence and Biotechnology Will Create the Medicines of the Future

  • Brian S. Hilbush(Author)
  • 2021(Publication Date)
  • Wiley

    (Publisher)

  7 To study gene regulation and function in eukaryotes, the CRISPR-Cas system was redesigned with the ability to turn genes on and off with transcriptional modifiers and also engineered with the ability to perform gene knockouts and knock-ins. The bio-tech tools of the CRISPR era are versatile, inexpensive, and even more democratized than their recombinant DNA ancestors, especially when Prime editors Base editors CRISPR/Cas9 + HDR CRISPR/Cas9 + NHEJ Gene augmentation Gene replacement time precision Zinc finger nucleases 2019 2020 2016 2013 Translocases Recombinases 2000s 1990s TALENs Gene cloning 1980s Figure 4.1: Biotechnology tools and the acceleration into an era of precision genome engineering The tools of recombinant DNA technology opened the door to the possibility of genetic manipulation of genes, traits, and organisms. The early tools and techniques (1980–1990s) could recombine and transfer gene segments into cells, including embryos, but lacked precision. Second-generation biotechnology tools based on CRISPR-Cas led to a dramatic shift in precision due to the nature of programmable nucleases. Continued research and development to engineer more efficient and precise editing technologies has led to dramatic leaps in the breadth of genomic edits possible with relatively few components: a programmable RNA guide and a multifunc-tional protein built on a Cas scaffold that enables high precision genome editing for an array of applications. TALENs: transcription activator-like effector nucleases; ZFNs: zinc finger nucleases; NHEJ: non-homologous end joining; HDR: homology-directed repair. Chapter 4 ■ Gene Editing and the New Tools of Biotechnology 149 it comes to transgenic animal applications.

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  Function and Evolution of Repeated DNA Sequences

  • Guy-Franck Richard(Author)
  • 2023(Publication Date)
  • Wiley-ISTE

    (Publisher)

  ecologists. They can be used to partially reconstruct the history of past infections, identify MGEs and their hosts, and thus gain a better understanding of the evolutionary dynamics of microbial populations and their parasites over time in various environments.

  8.4. An adaptive immune system

  8.4.1. A three-stage immune response

  The molecular mechanisms involved in the immune response mediated by CRISPR-Cas differ significantly depending on the type of system. Nevertheless, the general operating principle is relatively stable: following a primary infection, CRISPRs integrate fragments of the invasive agent as spacers; they are then transcribed and matured into small RNA molecules that will guide Cas proteins to specifically bind and cleave complementary foreign nucleic acids. This immune response can thus be broken down into three distinct stages: adaptation, expression and interference (see Figure 8.5 ).

  During the adaptation phase, the adaptation complex comprising the Cas1 and Cas2 proteins binds to an exogenous DNA molecule, such as that of a phage infecting the cell. This complex introduces two double-strand breaks in the foreign DNA, at the protospacer, generally upon recognition of a short motif (2–7 nt) known as PAM (protospacer-adjacent motif). In some cases, invasive DNA fragmentation may be achieved by RecBCD (when replication fork is stalled) or by restriction enzymes (from restriction-modification (RM) systems) present in the genome. The resulting fragment is then inserted at the 5’ end of the CRISPR at the leader sequence and becomes a new spacer. The leader sequence and a single repeat are required during this stage. The presence of recognition sites, both in the leader sequence and in the repeat, located within 10 bp of the integration site makes it possible to direct the adaptation complex to this precise position (Yosef et al. 2012; Wei et al. 2015). The CRISPR array is then repaired by the cellular repair machinery, resulting in the duplication of the proximal repeat. Some CRISPR-Cas systems use an alternative adaptation mechanism, that is, the acquisition of spacers from the RNAs of the invasive element via the action of reverse transcriptases (RTs) that reverse-transcribe viral RNA to DNA, genetic information that can then be integrated into the CRISPR array under the action of the adaptation complex. These RTs are generally encoded by the system itself and are often fused to cas1

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  Genome Editing in Drug Discovery

  • Marcello Maresca, Sumit Deswal, Marcello Maresca, Sumit Deswal(Authors)
  • 2022(Publication Date)
  • Wiley

    (Publisher)

  Cas9 is a dual RNA-guided DNA endo-nuclease required for conferring immunity in type II systems (Barrangou et al. 2007; Gasiunas et al. 2012; Jinek et al. 2012). Apart from crRNA (required by any other CRISPR system to recognize target molecule), Cas9 also requires trans-activating crRNA (tracrRNA), a noncoding RNA that coordinates the processing of crRNA and whose hairpin Cas9 binds to (Deltcheva et al. 2011). Once bound to crRNA:tracrRNA complex, Cas9 identifies target DNA through PAM recognition, and base pairing between the crRNA and the target DNA (Figure 3.5a). If sufficient complementarity is present, S. pyogenes Cas9 generates a blunt DNA double-stranded break (DSB) 3 bp upstream of the PAM through concerted activity of its RuvC and HNH domains (Jinek et al. 2012). Similarly to other systems, cleavage by Cas9 initiates further degradation by the host machinery, neutralizing the invading genome (Figure 3.5a). Detailed structural and biochemical studies of SpyCas9 protein have revealed a bilobed structure with the crRNA and target DNA accommodated in the central cleft. Cas9:crRNA:tracrRNA complex recognizes the PAM site via interaction of the GG dinucleotides and the conserved amino-acids of the C-terminal domain. Recognition of the correct PAM leads to a local unwinding of the DNA duplex, allowing the crRNA to pair with a 10–12 nt long seed sequence of the target strand (Anders et al. 2014; Sternberg et al. 2014). Successful pairing accompanied by further conformational changes prompts further invasion of the crRNA, forming a stable R-loop across the full length of crRNA (Jiang et al. 2016a; Mekler et al. 2017). This in turn induces another set of complex conformational changes, where the movement of the HNH domain leads to activa-tion of the RuvC domain, allowing coordinated cleavage of the target and nontarget strand, respectively (Sternberg et al. 2015; Raper et al. 2018).

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