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

Luis María Vaschetto

• Publisher: CRC Press
• Year: 2022
• Print ISBN: 9780367542870
• eBook ISBN: 9781000540833
• Edition: 1
• Topic: Mathematics
• Subtopic: Genetics in Medicine
• Index: Mathematics

The CRISPR-Cas9 genome-editing system is creating a revolution in the science world. In the laboratory, CRISPR-Cas9 can efficiently be used to target specific genes, correct mutations and regulate gene expression of a wide array of cells and organisms, including human cells. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases is a unique reading material for college students, academicians, and other health professionals interested in learning about the broad range of applications of CRISPR/Cas9 genetic scissors. Some topics included in this book are: the role of the CRISPR/Cas9 system in neuroscience, gene therapy, epigenome editing, genome mapping, cancer, virus infection control strategies, regulatory challenges and bioethical considerations.

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*

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

²Department 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).

Figure 1 Timeline progression and development of CRISPR and a brief description of fundamental discoveries. In 1987, during a study focused on alkaline phosphatase loci in E. coli, the first observation of invariably repeated sequences flanking specific sequences, later described as CRISPR arrays (in 2002 (Jansen et al. 2002)), was made by Ishino et al. (Ishino et al. 1987). Later, CRISPR sequences were discovered in other microorganisms (Mojica et al. 2000), and in 2005 foreign sequences (spacers) from phages and plasmids were identified in CRISPR (Bolotin et al. 2005, Haft et al. 2005). The bacterial “immune” function (Marraffini and Sontheimer 2008) of CRISPR, and the crRNAs that serve as gRNAs for DNA targeting (Brouns et al. 2008) are discoveries made in the following years. Between 2009-2011, CRISPR/Cas system to cleave RNA molecules (Hale et al. 2009), the specific DSB DNA cleavage (Garneau et al. 2010), and tracrRNA-crRNA structure associated with Cas9 were first described (Deltcheva et al. 2011). In 2012, another major breakthrough was made, as two separate studies indicated that CRISPR system operates as RNA-guided endonucleases (Gasiunas et al. 2012), respectively, that the Cas9 are reprogrammable and this system could potentially serve as a tool for genome-editing (Jinek et al. 2012). Another breakthrough is represented by the transfer of this technology from prokaryotes to eukaryotic cells in 2013, and in 2014 the first sgRNA libraries were used for genome-wide screening using Cas9 (Cong et al. 2013, Mali et al. 2013a, Wang et al. 2014, Shalem et al. 2015). Recent studies proved the efficiency of CRISPR/Cas systems in cancer biology (Sanchez-Rivera and Jacks 2015), and embryonic stem cells systems (Jin and Li 2016). In 2018, the first gene editing of HIV-1 co-receptors using CRISPR was made (Allen et al. 2018). In 2019, CRISPR/Cas9 was used to control genetic inheritance in mice (You et al. 2019), and in 2020, it was the first time when CRISPR was used as a genetic scissor directly administered into a patient (Ledford 2020).

Figure 2 Mechanism of action in CRISPR/Cas9 genome-editing system (after Chira et al. (Chira et al. 2017)). The discovery of the original CRISPR/Cas9 system in bacteria facilitated its transformation into a genome-editing tool. The tracr-RNA-crRNA forming a dual-RNA hybrid structure serving as gRNA can be inserted into the target cell, directed towards the nucleus. Then the cas9 gene is transcribed and exported outside the nucleus and translated into protein (Cas9). The interaction between the gRNA and the functional Cas9 protein generates a ribonucleic-protein effector complex able to target the gDNA (genomic DNA) at a specific locus, nearby to the PAM sequence. DSBs are then introduced, and the restoration of these brakes can be performed either through NHEJ (which increases the possibility of introducing indel mutations), or via HDR (which requires the presence of a donor DNA).

The bacterial origin of CRISPR/Cas

The discovery of CRISPR/Cas nucleases system

The coexistence of prokaryotes and viruses generated various defense mechanisms such as restriction modifications, toxin-antitoxin systems, and, in the later years, CRISPR/Cas systems were discovered (Labrie et al. 2010). The discovery of CRISPR as a component of the prokaryotic “immune” system, and the repurposing of this system as a genome editing tool, determined a broad use of this technology in molecular biology applications, making it one of the most used technologies in active research in biology (van Soolingen et al. 1993, Bolotin et al. 2005, van der Oost et al. 2009, Mali et al. 2013a, Cho et al. 2013) (Figure 1). Even though the first CRISPRs were observed decades ago in bacteria (Ishino et al. 1987), and subsequent studies revealed the presence of CRISPRs in archaea (Mojica et al. 1993), it was only in the early 2000s that researchers discovered the sequence similarities between the viruses, bacteriophages, and plasmids and the spacer regions in CRISPR, managing to uncover the defense function of CRISPR (Mojica et al. 2005, Bolotin et al. 2005). Independent parallel studies revealed a set of genes associated with CRISPR, consequently named cas (CRISPR-associated), and, in 2008, Marakova et al. suggested the existence of a CRISPR/Cas complex that acts as an acquired immune system to protect the bacterial cell against invading phages or other exogenous genetic material (Jansen et al. 2002, Makarova et al. 2006).

Components of the CRISPR/Cas system

CRISPR/Cas system ensures the immunity of the bacteria in three steps that require target recognition and cleavage (Barrangou and Marraffini 2014, Sorek et al. 2013). The first step is the adaptation, which allows the copy and paste of the foreign nucleic acids into the ‘spacers’ of the CRISPR arrays, thus providing acquired resistance against the invading phage (Barrangou et al. 2007). The next step includes the biogenesis of the crRNA (expression stage), in which the small interfering RNAs are generated through transcription and further processing. In the interference stage, the gRNA heads the Cas enzymes to cleave the DNA (Marraffini and Sontheimer 2008). The CRISPR-Cas system can be classified into two main categories according to the effectors: first, where all functionalities in the effector complexes are carried out using a protein, and second, the multi-unit effector complexes (Shmakov et al. 2017). These two classes are further divided into six types of CRISPR-Cas systems according to the presence of the signature genes. In type I systems, the signature protein is Cas3, in which the cleavage of the external DNA is carried out by the nuclease and helicase domains, and the recognition of the target sequence is made by the multi-protein-crRNA complex Cascade. In type II systems, the unique protein required for the interference is Cas9 (signature protein). In type III systems, the signature gene is cas10, a gene encoding a multidomain protein (Cas10) that is assembled into an interference complex needed for the identification and cleavage of the target sequence. Type IV systems are usually not linked to a CRISPR array. The effector complex is represented by Csf1 (csf1 being considered the signature gene) and the other two proteins encoded by cas genes (Makarova and Koonin 2015). Type V systems comprise a unique Cas9-like nuclease, which can be Cpf1, C2c1, or C2c3 according to the subtype of the CRISPR/Cas type V system (Shmakov et al. 2017, Zetsche et al. 2015). In type VI systems, the protein C2c2 with two HEPN RNase domains is the signature protein (Shmakov et al. 2017). All these systems are classified as either Class1 systems (Type I, III, IV) as they have a multi-subunit effector or as Class 2 systems (Type II, V, VI) as they are characterized by a single-subunit effector (Shmakov et al. 2017, Makarova and Koonin 2015). Table 1 ill...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Preface
• Table of Contents
• 1. The CRISPR/Cas9 Genome-editing System: Principles and Applications
• 2. Recent Applications of CRISPR-Cas9 in Genome Mappingand Sequencing
• 3. Gene Editing and Genetic Disorders: Ethical and Legal Concerns
• 4. Political, Regulatory and Ethical Considerations of the CRISPR/CasGenome Editing Technology
• 5. CRISPR/Cas and Gene Therapy: An Overview
• 6. CRISPR and Cancer: From Bench to Clinic
• 7. The Advances of the CRISPR/Cas9 Technology for CorrectingGenetic Disorders: Using Sarcoma as a Model System
• 8. CRISPR/Cas9 Technology as a Strategy Against Viral Infections
• 9. CRISPR/Cas9-based Genome and Epigenome Editing inNeuroscience Research
• 10. Non-viral Alternatives to Delivery of CRISPR-Cas GeneEditing Components
• 11. CRISPR Medicine: Advance, Progress and Challenges
• Subject Index