Genome Engineering via CRISPR-Cas9 System

Genome Engineering via CRISPR-Cas9 Systems presents a compilation of chapters from eminent scientists from across the globe who have established expertise in working with CRISPR-Cas9 systems. Currently, targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative simplicity to which they can be designed, used and applied. However, it is not easy to find relevant information gathered in a single source. The book contains a wide range of applications of CRISPR in research of bacteria, virus, algae, plant and mammalian and also discusses the modeling of drosophila, zebra fish and protozoan, among others.

Other topics covered include diagnosis, sensor and therapeutic applications, as well as ethical and regulatory issues. This book is a valuable source not only for beginners in genome engineering, but also researchers, clinicians, stakeholders, policy makers, and practitioners interested in the potential of CRISPR-Cas9 in several fields.

• Provides basic understanding and a clear picture on how to design, use and implement the CRISPR-Cas9 system in different organisms
• Explains how to create an animal model for disease research and screening purposes using CRISPR
• Discusses the application of CRISPR-Cas9 systems in basic sciences, biomedicine, virology, bacteriology, molecular biology, neurology, cancer, industry, and many more

• Language: English
• Publisher: Academic Press
• Release date: Feb 18, 2020
• ISBN: 9780128181416

Book preview

Genome Engineering via CRISPR-Cas9 System

Editors

Vijai Singh

Department of Biosciences, Indrashil University, Mehsana, Gujarat, India

Pawan K. Dhar

School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

Table of Contents

Cover image

Title page

Copyright

Dedication

Contributors

About the editors

Foreword

Preface

Acknowledgments

Chapter 1. An introduction to genome editing CRISPR-Cas systems

1.1. Introduction

1.2. History and classification of CRISPR-Cas systems

1.3. Milestones in the CRISPR-Cas systems

1.4. Development of CRISPR-CAS9 system for genome editing

1.5. Recent developments in CRISPR interference platform

1.6. Conclusion and future remarks

Chapter 2. Evolution and molecular mechanism of CRISPR/Cas9 systems

2.1. Introduction

2.2. Evolution of CRISPR/Cas9 systems

2.3. Classification of CRISPR/Cas systems

2.4. Molecular mechanism of CRISPR/Cas-mediated defense systems

2.5. Application of CRISPR/Cas9 systems

2.6. Conclusions

Chapter 3. Exploring the potential of CRISPR-Cas9 for the removal of human viruses

3.1. Introduction

3.2. CRISPR-Cas9 system as an antiviral agent

3.3. CRISPR delivery in mammalian cells

3.4. Challenges to the use of CRISPR-CAS9 as therapy

3.5. Conclusion and future perspective

Chapter 4. Programmable removal of bacterial pathogens using CRISPR-Cas9 system

4.1. Introduction

4.2. Mechanism of CRISPR-Cas systems

4.3. Application of CRISPR-Cas9 system as an antimicrobial agent

4.4. Bacteriophage engineering to extend the host range

4.5. Conclusion and future remarks

Chapter 5. Targeted genome editing using CRISPR/Cas9 system in fungi

5.1. Introduction

5.2. Genome editing in yeasts

5.3. Genome editing in filamentous fungi

Chapter 6. CRISPR-Cas9 system for fungi genome engineering toward industrial applications

6.1. Introduction

6.2. Challenges in editing fungal genome

6.3. Industrial applications of CRISPR-Cas9 methods in fungi genome editing

6.4. Implementations of the CRISPR-Cas9 in fungi

6.5. CRISPR-based gene regulation in fungi

6.6. CRISPR-Cas9 a novel approach for biological control

6.7. Further developments required for fungi genome editing

6.8. Conclusion and future prospects

Chapter 7. Development and challenges of using CRISPR-Cas9 system in mammalians

7.1. Introduction

7.2. Principle mechanism behind CRISPR-Cas9 mediated gene editing

7.3. Delivery of CRISPR-Cas9 component

7.4. Recent development and applications of CRISPR-Cas9 for human and mammalian diseases

7.5. Key issues and challenges

7.6. Conclusions and future remarks

Chapter 8. CRISPR-Cas9 system a mighty player in cancer therapy

8.1. Introduction

8.2. Functional characterization of cancer-related genes by conventional methods

8.3. Involvements of the non-coding region of the human genome in a certain type of cancers could give a novel therapeutic targets

8.4. Challenges and advancement needed in CRISPR-Cas9 method for cancer treatments

8.5. CRISPR-Cas9 and the future of cancer therapy

Chapter 9. CRISPR-Cas9 for therapy: the challenges and ways to overcome them

9.1. Introduction

9.2. CRISPR-Cas9 as a drug

9.3. A match made in heaven; iPSC and CRISPR-Cas9

9.4. Ex-vivo versus in-vivo editing

9.5. Bench-to-bedside challenges

9.6. Conclusion

Chapter 10. Engineering of Cas9 for improved functionality

10.1. Introduction

10.2. Cas9 variants with altered nuclease activity

10.3. Cas9 variants with improved PAM specificity

10.4. Switchable Cas9

10.5. Inducible Cas9

10.6. gRNA editing

10.7. Other CRISPR-associated endonucleases

Chapter 11. The current progress of CRISPR/Cas9 development in plants

11.1. Introduction

11.2. Mechanism of Crispr/Cas9

11.3. Multiplex Crispr/Cas9

11.4. Metabolic engineering in plants using Crispr/Cas9

11.5. Crispr/Cas9 mediated live cell imaging

11.6. Non-transgenic plants through CRISPR/Cas9

11.7. Conclusions and future remarks

Chapter 12. Fruit crops improvement using CRISPR/Cas9 system

12.1. Introduction

12.2. Nutritional aspects of fruit crops

12.3. Genome editing as a tool towards fruit crop improvement

12.4. Crispr/Cas9 system: a tool for improving stress tolerance in fruit crops

12.5. Challenges pertaining to fruit crop improvement via Crispr/Cas9 technology

12.6. Conclusion and future perspective

Chapter 13. CRISPR/Cas9 engineered viral immunity in plants

13.1. Introduction

13.2. Plant viruses and existing virus control strategies

13.3. Gene editing with CRISPR-Cas system

13.4. CRISPR-Cas-mediated viral resistance through PDR approach

13.5. CRISPR-Cas mediated viral resistance by interfering host encoded genes

13.6. Conclusion and future perspectives

Chapter 14. Genome engineering in medicinally important plants using CRISPR/Cas9 tool

14.1. Introduction

14.2. Designing of gRNA and vectors construction

14.3. CRISPR-Cas9 construct delivery methods into plant cells

14.4. Pathway engineering using CRISPR-Cas9

14.5. Editing in hairy roots of medicinal plants for producing secondary metabolites

14.6. Future perspective of genome editing in medicinal plants

Chapter 15. Genome editing of algal species by CRISPR Cas9 for biofuels

15.1. Introduction

15.2. Genetic engineering of algae

15.3. CRISPR

15.4. CRISPR over random mutagenesis and RNAi

15.5. Target pathways for development of microalgae as biofuel feedstocks

15.6. CRISPR-Cas9 in microalgae

15.7. CRISPR workflow in microalgae

15.8. Conclusion, challenges and future remarks

Chapter 16. Development and use of CRISPR in industrial applications

16.1. Historical perspectives

16.2. Development of CRISPR based technologies

16.3. Design tools for CRISPR-Cas9 based genome editing

16.4. Industrial products developed using CRISPR

16.5. Conclusion

Chapter 17. Functional understanding of CRISPR interference: its advantages and limitations for gene silencing in bacteria

17.1. Introduction

17.2. Gene silencing by CRISPRi in bacteria

17.3. Advantages and limitations of CRISPRi in bacteria

17.4. Concluding remarks

Chapter 18. Genome engineering in insects: focus on the CRISPR/Cas9 system

18.1. Introduction

18.2. Tools used for genome engineering

18.3. Genome engineering in insects using CRISPR/Cas9

18.4. Targeting efficiency and off-target effects of CRISPR/Cas9

18.5. Genome engineering in insects using ZFN

18.6. Genome engineering in insects using TALEN

18.7. Gene drive

18.8. Ethical concerns of genome editing

18.9. Conclusion

Chapter 19. Recent progress of CRISPR-Cas9 in zebra fish

19.1. Introduction

19.2. Methods of genome editing

19.3. CRISPR-Cas9 system for genome editing of zebra fish

19.4. Applications of CRISPR-Cas9 in zebrafish

19.5. Conclusion and future remarks

Chapter 20. CRISPR: a revolutionary tool for genome engineering in the protozoan parasites

20.1. Introduction

20.2. Limitations in genome engineering of apicomplexan parasites

20.3. Gene editing tools for studying protozoan parasites

20.4. Concluding remarks

Chapter 21. Emergent challenges for CRISPR: biosafety, biosecurity, patenting, and regulatory issues

21.1. Introduction

21.2. Biosafety

21.3. Biosecurity

21.4. Patenting CRISPR technologies and products

21.5. Regulatory issues with CRISPR products

21.6. Conclusions and future remarks

Appendices

Glossary

Author Index

Subject Index

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

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Dedication

Dedicated to my beloved wife Pritee and lovely kids Aaradhya and Ayush for being source of my inspiration and strength.

Vijai Singh

Dedicated to my adorable wife Sunita and lovely kids Riya and Shrea for being the source of my strength.

Pawan K. Dhar

About the editors

Dr. Vijai Singh is an Associate Professor in the Department of Biosciences, School of Science at Indrashil University, Mehsana, Gujarat, India. Prior to this, Dr. Singh was a postdoctoral fellow at Institute of Systems and Synthetic Biology, France and School of Energy and Chemical Engineering at Ulsan National Institute of Science and Technology, South Korea. Dr. Singh has served as an Assistant Professor in the Department of Biotechnology at Invertis University, India and Department of Biological Sciences and Biotechnology, Institute of Advanced Research, India. Dr. Singh received his Ph.D. in Biotechnology from Dr. A.P.J. Abdul Kalam Technical University/National Bureau of Fish Genetic Resources, Lucknow, India with a research focus on the development of molecular and immunoassay for Aeromonas hydrophila. Dr. Singh has designed and characterized a number of synthetic oscillators, gene networks, lycopene pathway, MAGE and CRISPR-Cas9 system in Escherichia coli. Currently, Dr. Singh's laboratory focuses on the construction of a novel biosynthetic pathway for the production of pigments, chemicals, and fuels. Additionally, Dr. Singh's laboratory works on developing CRISPR-based platform for disease diagnosis and eradication of MDR pathogens. Dr. Singh has published 72 articles, 19 chapters, and 1 book. Dr. Singh serves as a member of the editorial board and reviewer of a number of peer-reviewed journals.

Prof. Pawan Kumar Dhar is the Dean of School of Biotechnology, Jawaharlal Nehru University, New Delhi and heads the synthetic biology group. Prior to this, he held scientific positions at RIKEN Institute, Keio University and Kyoto University (Japan), Bioinformatics Institute, (Singapore) and Manipal University (India). Prof. Dhar received his Ph.D. in 1993 from Banaras Hindu University (Varanasi) for his work on Human Genetics. Currently, Prof. Dhar's lab works on making functional genes and proteins from the non-expressing genome finding applications in health, energy, and environment. Prof. Dhar serves in the external board of referees for European Science Foundation and Indian Government science funding agencies.

Contributors

Ali Samy Abdelaal

Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

Department of Genetics, Faculty of Agriculture, Damietta University, Damietta, Egypt

Sundaram Acharya

CSIR-Institute of Genomics & Integrative Biology, New Delhi, India

Academy of Scientific & Innovative Research, New Delhi, India

Nisheeth Agarwal,     Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India

Anshu Alok,     University Institute of Engineering & Technology, Panjab University, Chandigarh, India

Takayuki Arazoe,     Tokyo University of Science, Department of Applied Biological Science, Faculty of Science and Technology, Chiba, Japan

Praveen Awasthi,     National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India

Abhisheka Bansal,     School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Purva Bhalothia,     Dr. B. Lal Institute of Biotechnology, Jaipur, Rajasthan, India

Gargi Bhattacharjee

Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India

Kul Bhushan,     Division of Fruits and Horticultural Technology, ICAR-Indian Agricultural Research Institute, New Delhi, India

Darren Braddick,     Department of R&D, Cementic S.A.S., Genopole, Paris, France

Stanislaus Antony Ceasar

Functional Genomics and Molecular Imaging Lab, University of Liege, Liege, Belgium

Division of Biotechnology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India

Debojyoti Chakraborty

CSIR-Institute of Genomics & Integrative Biology, New Delhi, India

Academy of Scientific & Innovative Research, New Delhi, India

Dharmendra Kumar Chaudhary,     Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Eira Choudhary

Symbiosis School of Biomedical Sciences, Symbiosis International University, Pune, Maharashtra, India

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India

Jaspreet Kaur Dhanjal,     Department of Biochemical Engineering and Biotechnology, DBT-AIST International Laboratory for Advanced Biomedicine (DAILAB), Indian Institute of Technology Delhi, New Delhi, India

Pawan K. Dhar,     School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

Nisarg Gohil

Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India

Radhika Gupta,     Daulat Ram College, University of Delhi, India

V. Edwin Hillary,     Division of Biotechnology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India

S. Ignacimuthu

Division of Biotechnology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India

Xavier Research Foundation, St. Xavier's College, Palayamkottai, Tamil Nadu, India

Prateek Jain,     Department of Biology, University of North Carolina, Chapel Hill, NC, USA

Navneet Kaur

National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India

Department of Biotechnology, Panjab University, Chandigarh, India

Khushal Khambhati,     Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Ankit Kumar,     Department of Genetics & Plant Breeding, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, India

Shashi Kumar,     Metabolic Engineering Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India

Jitesh Kumar,     Center of Innovative and Applied Bioprocessing, Mohali, Punjab, India

Ariel Kushmaro,     Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel

Ajitesh Lunge,     Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India

Souvik Maiti

CSIR-Institute of Genomics & Integrative Biology, New Delhi, India

Academy of Scientific & Innovative Research, New Delhi, India

CSIR-National Chemical Laboratory, Pune, India

Siddharth Manvati,     School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

Osamu Mizutani,     Univeristy of the Ryukyus, Department of Bioscience and Biotechnology, Faculty of Agriculture, Okinawa, Japan

Balasubramanian C. Muthubharathi,     Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India

Happy Panchasara,     Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Shreya Patel,     Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Dharmendra Pratap,     Department of Genetics & Plant Breeding, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, India

Rina Fanny Ramarohetra,     Independent Researcher, Paris, France

Lakkakula Satish

Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel

The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Bergman Campus, Beer Sheva, Israel

Sasanala Shamili,     The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Bergman Campus, Beer Sheva, Israel

Manish Sharma,     School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Rishabh Sharma,     Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India

Sandeep Kumar Singh,     Indian Scientific Education and Technology Foundation, Lucknow, Uttar Pradesh, India

Vijai Singh

Department of Biological Sciences & Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar, Gujarat, India

Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India

Yaron Sitrit,     The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Bergman Campus, Beer Sheva, Israel

Durai Sundar,     Department of Biochemical Engineering and Biotechnology, DBT-AIST International Laboratory for Advanced Biomedicine (DAILAB), Indian Institute of Technology Delhi, New Delhi, India

Amita Tanwar,     Metabolic Engineering Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India

Siddharth Tiwari,     National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India

Santosh Kumar Upadhyay,     Department of Botany, Panjab University, Chandigarh, India

Dhvani Sandip Vora,     Department of Biochemical Engineering and Biotechnology, DBT-AIST International Laboratory for Advanced Biomedicine (DAILAB), Indian Institute of Technology Delhi, New Delhi, India

Kalpesh Yajnik,     Dr. B. Lal Institute of Biotechnology, Jaipur, Rajasthan, India

Syed Shams Yazdani

Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

Foreword

I am delighted to accept the invitation from Dr. Vijai Singh and Prof. Pawan K. Dhar in order to provide some introductory statements to Genome Engineering via CRISPR-Cas9 System, a timely volume on the rapidly evolving field of CRISPR-Cas9 system applied to genome engineering.

The ground-breaking advances in genome editing and regulation that the CRISPR-Cas9 technology has introduced in just a few years have revolutionized our ability to perform genome engineering. CRISPR-Cas9 is simple, sensitive, specific, efficient and easy to use for genome editing. CRISPR-Cas9 is used for creating animal models for the understanding of the molecular mechanism, removal of viruses, bacteria, fungi and other pathogens in animals as well as plants, repairing disease-causing defective genes of neurological, hereditary, cancer, diabetes, and for increased production of chemicals, drugs, metabolites, biofuels and many more. Notably, the CRISPR-Cas9 technology has been modified and expanded into applications such as strain engineering, metabolic engineering, gene activation, repression, screening, tagging, imaging, etc.

This comprehensive book contains 21 chapters with various aspects of CRISPR biology from basics, discovery, history, innovation, practice, and applications. The book covers the use of technology in a wide range of organisms including bacteria, fungi, algae, viruses, Drosophila, protozoan, zebrafish, mosquitoes, mammals and many more. Applications and topics resourced in the chapters range from biomedical to industrial; from immunity and cell line development to plant, algae and biofuels. Chapters are written by scientists from across the globe with a renowned and established expertise on CRISPR-Cas systems.

I am pleased to recognize the valuable efforts of Dr. Vijai Singh and Prof. Pawan K. Dhar, who together brought out an excellent volume through the world's-leading publisher in science-Elsevier. I believe that this volume constitutes an excellent and informative text on genome engineering using CRISPR-Cas9 system with a simple and easy to understand format. It is a great pleasure to announce this book that will render CRISPR-Cas knowledge to the beginners in genome engineering, researchers, students and scientists, clinicians, stakeholders, policymakers, and practitioners, among many others.

Pablo Carbonell, PhD

Senior Staff Scientist

Manchester Institute of Biotechnology

The University of Manchester, United Kingdom

Preface

Targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative ease with which the toolkit can be designed and applied to generate desired behaviours. In the past decade, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been customized for making double-stranded breaks (DSBs) at selected target sites. Despite significant scientific advances, designing a target-specific binding nuclease is still challenging, tedious, laborious, and expensive. Due to this reason, developing a more precise and simple method to edit genomes became inevitable. As a result of several interesting and unconnected findings moving through a tortuous route, a new technology called CRISPR-Cas9 was born. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated proteins (Cas) were found to exist as RNA-guided adaptive immune system of prokaryotes, conferring protection against phages and promiscuous plasmids. CRISPR-Cas systems are mainly divided into six types (I–VI). CRISPR systems are RNA-guided that can bind specifically to a target site and then create double-stranded breaks subsequently repaired by non-homologous end joining or the homology-directed repair pathway.

By 2012, the CRISPR-Cas9 system was proven to be a preeminent technology for targeted genome editing, acting as a simple, rapid, and cost-effective solution. In the last few years, a number of studies have been demonstrated that have used CRISPR-Cas9 as a groundbreaking technology to create animal models, treat hereditary and neurological diseases, remove bacterial pathogens and viruses, develop new strains, activate, repress, screen, tag or imagine genes and so on. CRISPR-Cas9 is often used to knock out competitive pathways in order to increase the carbon flux towards the production of targeted chemicals.

This book brings together contribution from scientists from across the globe with hands-on experience in the CRISPR-Cas9 system. This book covers a wide range of topics from bacteria, viruses, algae, plants, mammals and other model organisms including Drosophila, zebrafish, protozoan, and many more. Across the functional spectrum, the book covers topics related to diagnostics, therapeutics and ethical as well as regulatory issues.

The book has been designed to benefit researchers, students and scientists, clinicians, stakeholders, policymakers, and so on. The work presented may be used to develop a genome editing course for students. This book is a compilation of 21 chapters written by eminent scientists from seven countries including Belgium, Egypt, France, India, Israel, Japan, and the USA. It is our hope that this book not only provides a genome editing guide but also triggers unanswered questions. Though monumental efforts have been invested to make this book user-friendly, we are aware that the first version always comes with bugs. We would be happy to receive suggestions to improve the book further.

Vijai Singh and Pawan K. Dhar

Acknowledgments

Vijai Singh

I am delighted to thank all the authors for their excellent contributions and reviewers for their comments as well as suggestions to improve the quality of the chapter. I would like to express my sincere gratitude and deep appreciation to Dr. J.S. Yadav, Director (Research), Indrashil University, India who gave me outstanding support and motivation to complete this book. I would also like to give many thanks to Prof. Pawan K. Dhar (Co-Editor of this book) who gave me outstanding personal and professional support as well as inspiration to finish this book.

I would like to thank Rafael Teixeira (Acquisitions Editor), Samuel Young (Editorial Project Manager) and Kavitha Balasundaram (Copyrights Coordinator) from Elsevier for their excellent management of this project and anonymous reviewers for their recommendation for this book. I also thank Mark Rogers (Book Designer) for designing the cover page and incorporating my suggestions.

I greatly appreciate the support of my students Nisarg Gohil and Gargi Bhattacharjee, whose discussion and comments helped to shape this book. I thank Vimal C. Pandey, Satya Prakash, and those whose names do not feature here but have directly or indirectly helped me in shaping this project.

I wish to express my gratitude to my beloved wife, Pritee Singh for her endless support, patience, and inspiration. I thank my kids Aaradhya and Ayush, who missed me during this project.

I would like to warmly thank faculties and staffs of Indrashil University for providing a great working environment. Last but not least, my sincere thanks to GOD for his supreme POWER, and endowing me to live with joy and victory in the shape of the book.

Pawan K. Dhar

Ignorance is necessary for the existence of knowledge. A conscious walk in the space of the unknown is exciting and fulfilling.

The year 2012 was a major innovative landmark in genome engineering when Dr. Jennifer Doudna, Dr. Emmanuelle Charpentier, Dr. Feng Zhang, and many other amazing scientists developed techniques to remove and insert DNA at predefined locations. Their work had such an impact that the whole scientific community got rebooted towards new innovative pathways.

At the outset, I warmly acknowledge the incredible work of genome editing pioneers who showed new ways of engineering genomes. Second, I sincerely thank the countless PIs, Ph.D. students, Post-Docs, technicians and interns who worked hard to innovate in this area and sustained the momentum of genome editing, leading to various applications.

To assemble a large body of published work into an easily readable format for students, we needed a strong team that delivers quality output while retaining the readability for both novices and experts. I warmly thank my fellow Editor, Dr. Vijai Singh for his initiative in conceptualizing this book, contacting leading scientists and coordinating with Elsevier to build this wonderful work. My sincere thanks to our eminent colleagues for allocating their prime time towards this book, despite their pressing deadlines.

I wish to warmly thank JNU Administration for providing a great working environment. At a personal level, my sincere thanks to my family members, students, and friends for their kind support and motivation.

Chapter 1

An introduction to genome editing CRISPR-Cas systems

Vijai Singh     Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India

Abstract

A CRISPR-Cas system is an extensively studied defense mechanism that protects bacteria and archaea against invading bacteriophage and plasmids. Currently, it is being used for editing the genome in many organisms. CRISPR-Cas system is a key genome editing technology which is simple, cost-effective, specific and user-friendly. It requires the expression of Cas9 endonuclease, single guide RNA and PAM sequence. The Cas9-sgRNA complex binds and creates a double-stranded break (DSB) that is repaired by non-homologous end joining (NHEJ) or by the homology-directed repair (HDR) pathway. This allows the generation of indels, which in turn allows modification of target DNA sequences. More recently, a number of Cas versions have been discovered and developed for gene editing, imaging of genomic loci, gene regulation, diagnostics and many more. In this chapter, we highlight the recent progress, use and challenges of the CRISPR platform for a wide range of therapeutic and biotechnological applications.

Keywords

CRISPR-Cas9; Genome editing; sgRNA; PAM; DSB; Regulation; Therapy

1.1. Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems are RNA-mediated immune system in prokaryotes that protects them against bacteriophage and plasmids (Barrangou et al., 2007; Marraffini and Sontheimer, 2008; Horvath and Barrangou, 2010; Barrangou and Marraffini, 2014; McGinn and Marraffini, 2016). The Type II CRISPR-Cas9 system is extensively used for targeted genome editing in a number of organisms including bacteria, yeast and mammals (Jinek et al., 2012; Mali et al., 2013; Cong et al., 2013; Jiang et al., 2013; DiCarlo et al., 2013; Bikard et al., 2014; Jakočiūnas et al., 2015).

CRISPR-Cas9 technology has been developed and expanded for editing the genome of zebrafish (Hwang et al., 2013; Hisano et al., 2015; Liu et al., 2017; Cornet et al., 2018), Drosophila (Port et al., 2014; Ren et al., 2014), defective genes corrections (Long et al., 2014; Nelson et al., 2016; Guan et al., 2016) and for the removal of viruses such as HIV-1 (Ebina et al., 2013; Zhu et al., 2015), human papillomavirus (Kennedy et al., 2014), hepatitis B virus (Lin et al., 2014; Zhen et al., 2015) and the latent Epstein-Barr virus from human cells (Wang and Quake, 2014).

CRISPR-Cas9 has been repurposed to make CRISPR interference (CRISPRi) which is widely used in gene regulation, imaging loci, epigenetic modification and high throughput screening in a wide range of organisms (Qi et al., 2013; Bikard et al., 2013; Gilbert et al., 2013; Chen et al., 2013; Ma et al., 2015). The aim of this chapter is to present the recent developments and progress of CRISPR technology for therapeutic, biomedical and biotechnological applications.

1.2. History and classification of CRISPR-Cas systems

The CRISPRs era began in the 1980s when it was first discovered in Escherichia coli (Ishino et al., 1987). It was thought to be associated with a number of cellular functions such as replicon partitioning (Mojica et al., 1995), thermal adaptation (Riehle et al., 2001), DNA repair (Makarova et al., 2002) and rearrangements in the chromosome (DeBoy et al., 2006). It has been found that bacteria and archaea evolved CRISPR as an adaptive mechanism that protects them against incoming phage infection and horizontal plasmid transmission. Till date, CRISPR-Cas system has been found in 50% of bacterial and 90% of archaeal genomes (Horvath and Barrangou, 2010; Richter et al., 2012; Makarova et al., 2015; Singh et al., 2017; Hille et al., 2018). Currently, CRISPR is being extensively studied and used in basic and applied research. CRISPR-Cas systems are classified into two major classes (1 and 2), six types (I-VI), and 18 subtypes. All the CRISPR types have different signature protein, targeting DNA, RNA or both (Makarova et al., 2015; Hille et al., 2018).

Class 1 CRISPR–Cas systems form a comparatively larger group due to the presence of multiple proteins and crRNA effector complex molecule. It contains three CRISPR types i.e., type I, III and IV. Generally, the CRISPR-Cas systems work in the order of acquisition, RNA processing and interference (Singh et al., 2017). Cas1 and Cas2 are normally present in genome and it processes incoming DNA molecule in order to produce an active CRISPR-Cas system (Bhaya et al., 2011). In the second stage i.e. RNA processing, preCRISPR RNA (pre-crRNA) is transcribed from CRISPR locus via RNA polymerase and later endonucleases cleave pre-crRNA into the active form of CRISPR RNAs (crRNAs). It then proceeds for interference where crRNAs form a complex with Cas proteins and bind with the target region via complementary base pairing with high specificity for foreign DNA or RNA. Then it can degrade the DNA molecule for acquiring phage immunity (Cui et al., 2008; Brouns et al., 2008; Garneau et al., 2010).

All the classes, types and subtypes of CRISPR have been shown in Fig. 1.1. Class1 is more complex because of a wide range of multiple proteins involved for immunity. Type I contains a Cas3 protein that has an ssDNA (single-stranded DNA) nuclease and ATP dependent ligase for its activity (Nam et al., 2011; Chylinski et al., 2013). In brief, there are number of signature proteins involved in various type of CRISPR-Cas system such as type IA (Cas8a, Cas5), IB (Cas8b), IC (Cas8c), ID (Cas10d), IE (Cse1, Cse2), IF (Csy1, Cys2, Cys3), IU (GSU0054), type III (Cas10), IIIA (Csm2), IIIB (Cmr5), IIIC (Cas10 or Csx11), IIID (Csx10), and type IV (Csf1), IVA and IVB (unknown) (Makarova et al., 2011, 2015; Hille et al., 2018). Majority of Cas protein functions are yet to be determined but it can function with multiple associated proteins which allow the organism to gain resistance against infection.

Class 2 is important because it is quite simple and involves just a single functional protein. Currently, in Class 2, type II CRISPR-Cas9 system is recognized as a key technology for genome editing in a broad spectrum of organisms. It only targets the DNA molecule. In order to increase the use of CRISPR, type V and VI can be used that targets both DNA and RNA molecules. Type II contains Cas9 protein which has RuvC and HNH functional domains for generating a double-stranded break at target site in the presence of PAM (Protospacer adjacent motif, it is a 3–6 bases long nucleotide sequence). Type IIA comprises a Csn2 protein which is a ring-shaped DNA binding protein that is involved in the adaptation of type II CRISPR system. Type IIB contains Cas4 while for IIC, its associated signature protein and its function remains unknown and is yet to be determined. Type V contain Cpf1, C2c1 and C2c3 which have active RuvC domains but lacks HNH, therefore, these proteins can break single-strands and form a nick. Type VI (Cas13a, Cas13b, and Cas13c) has been recently discovered and it has RNA-guided RNase activity that can bind to DNA or RNA. This was recently used to develop an ultrasensitive diagnostic platform for detection of bacteria, virus, a mutation in the cancer-causing gene and many more (East-Seletsky et al., 2016; Myhrvold et al., 2018; Gootenberg et al., 2018; Khambhati et al., 2019). The focuses of synthetic biologists are high and with every new CRISPR discovery, the scope of CRISPR biology gets accelerated and widens.

Fig. 1.1  Classification of CRISPR-Cas systems and their signature genes. It has been classified mainly class 1 and 2 which have been categorized into type and subtypes of CRISPR systems ( Makarova et al., 2015 ).

1.3. Milestones in the CRISPR-Cas systems

Fig. 1.2 represents key milestones of CRISPR-Cas systems. The CRISPR era started with the first reports about the presence of CRISPR arrays in Gram-negative bacteria (Ishino et al., 1987). It has shown to play some important roles in a variety of cellular functions that include thermal adaptation, replicon partitioning, repair of DNA and chromosome rearrangement. Hermans et al. (1991) were the first to identify CRISPR arrays in Gram-positive bacteria and later Mojica et al. (1993) for the first time ever identified CRISPR arrays in archaea. Initially, the functionality of CRISPR was unknown; it was first reported by Mojica et al. (1995). By 2000, slowly and steadily a large number of CRISPR arrays came into consideration in bacteria and archaea (Mojica et al., 2000; Jansen et al., 2002). Jansen et al. (2002) first identified the CRISPR-associated (Cas) genes. This was the beginning of a real picture of a gene that could be involved in adaptive immunity of microorganisms. In the year 2005, many research groups identified the CRISPR spacers which are sequences homologous to those that are present in plasmid and bacteriophage (Bolotin et al., 2005; Pourcel et al., 2005; Mojica et al., 2005). In addition to this, it was also found that CRISPR-Cas systems have shown to play a defensive mechanism in bacterial cells (Mojica et al., 2005).

Fig. 1.2  History, development and key milestones of CRISPR-Cas systems.

Barrangou et al. (2007) first demonstrated the CRISPR-Cas systems that are associated with acquired immunity against phages. In addition to this, it was also reported that the CRISPR-Cas systems can interfere with horizontal plasmid transfer by targeting and degrading DNA (Marraffini and Sontheimer, 2008). It was described as the function of crRNA that was considered as a guide for CRISPR interference (Brouns et al., 2008). In a report, Deveau et al. (2008) emphasized that the protospacer sequences are important for CRISPR mediated bacteriophage immune response. The first ever demonstration of the CRISPR-Cas system proved that it could cut the DNA at a specific and precise location (Garneau et al., 2010). Later, trans-activating crRNA (tracrRNAs) were identified (Deltcheva et al., 2011). CRISPR-Cas system was transferred from two distinct bacterial species, i.e., from Streptococcus thermophilus to E. coli (Sapranauskas et al., 2011).

CRISPR-Cas system is a futuristic technology for genome editing. It was tested in vitro and was found to confer RNA guided genome editing capacity (Gasiunas et al., 2012; Jinek et al., 2012). Around the beginning of 2013, two landmark articles were published in "Science", which were a real breakthrough in genome editing technology. CRISPR-Cas9 was used for targeting the mammalian cells' genome (Mali et al., 2013; Cong et al., 2013). Subsequently, a number of research groups have globally developed the CRISPR-Cas9 system in many organisms for targeted genome editing (Shen et al., 2013; Wang et al., 2013; Yang et al., 2013). Ran et al. (2013) developed a Cas9n nickase for better target selection that considerably takes lesser time for genome editing. A complex of Cas9 along with sgRNA/apo-Cas9 was determined that could aid an additional layer of CRISPR based genome engineering (Jinek et al., 2014; Nishimasu et al., 2014). CRISPR based genomic libraries have been generated for genome-wide screening (Wang et al., 2014; Shalem et al., 2014). In 2015, CRISPR-Cas9 was used for Duchenne muscular dystrophy (DMD) disease for targeting the exons in the dystrophin gene by using single or multiplexed sgRNAs. It restored muscle strength in a mouse model allowing the consideration of CRISPR for future therapeutic applications (Ousterout et al., 2015).

Homology-independent targeted integration (HITI) has been developed using CRISPR. This could allow to knock in a gene of interest in both non-dividing and diving cell in vivo and in vitro (Suzuki et al., 2018). In 2017, another version of CRISPR, Cas13a started being used for RNA editing (Cox et al., 2017; Abudayyeh et al., 2017). Gootenberg et al. (2017) have developed a simple, ultrasensitive diagnostic platform ($0.61 per test) for detection of pathogenic bacteria, viruses and mutation in the cancer cell. In addition, it is possible to make a correction in the disease-causing gene of human embryos and to support this fact, a gene responsible for heart muscle thickening has already been modified (Ma et al., 2017). CRISPR has been used to insert a gene that can burn the fat of mice and pig. It could reduce up to 20% of fat resulting in a low-fat future animal (Zheng et al., 2017). In 2018, a Chinese Professor He Jiankui (Southern University of Science and Technology, Shenzhen) edited human baby embryos and targeted the CCR5 receptor gene for blocking the HIV entry and was successful in his attempts. For the proper treatment of disease, a pressing need has arisen for rapid diagnosis and monitoring. Therefore, CRISPR-based next generation simple, rapid, specific and ultrasensitive lateral flow diagnostic platform has been developed (Myhrvold et al., 2018; Gootenberg et al., 2018; Khambhati et al., 2019). CRISPR technologies are moving fast and have a great potential to solve societal and environmental issues and challenges. More innovation in CRISPR biology is yet to be explored in the near future toward many biotechnological applications.

1.4. Development of CRISPR-CAS9 system for genome editing

1.4.1. Microbial genome editing using CRISPR-Cas9 system

Currently, CRISPR-Cas9 system is being developed and used for targeted genome editing of pathogenic microorganisms for controlling infections and diseases. Besides this, it has been used for modifying the beneficial microorganisms for improving the production of metabolites, chemicals and biofuels. CRISPR-Cas9 has been used as an antimicrobial agent (Citorik et al., 2014; Bikard et al., 2014). The foundation for CRISPR-Cas9 was used for knocking out drug resistance gene, virulence factor and many more. Microbial targeted genome editing was performed by Jiang et al. (2013), where they developed a dual-RNA:Cas9 system whose specificity could be changed by altering nucleotides present in crRNA to make single and multiple changes. Two crRNAs were simultaneously used to generate multiplex mutagenesis and tested in Streptococcus pneumoniae and E. coli and found 100% efficiency along with 60 desired mutations.

In addition, Bikard et al. (2014) have developed a CRISPR-Cas9 system for sequence-specific removal of the targeted pathogen from a mixed population. They designed and constructed a staphylococcal vector by inserting Cas9 and tracrRNAs for biogenesis of the crRNA. They targeted the aph-3 gene (kanamycin resistance) and used the vector to specifically target and kill the pathogenic strain of Staphylococcus aureus while the non-pathogenic S. aureus population remained safe. In order to check the ability of multiplexing using CRISPR-Cas9, phagemids have been engineered with an array of CRISPR and were used to target either superantigen enterotoxin sek gene or a portion of mecA gene. This resulted in lethality of the strain (Bikard et al., 2014). Similarly, Citorik et al. (2014) designed CRISPR-Cas9 system that allows them to target a particular bacterial strain from a complex community. They used CRISPR to create a DSB in blaNDM-1 and blaSHV-18 strains. Those genes confer antibiotics resistance. CRISPR-Cas9 has also been transferred into E. coli to chromosomally target a gene and resultantly 1000-fold repression in the transformation efficiency was observed. A bacteriophage-mediated targeting of E. coli EMG2 in the mixed population was attempted and a significant reduction of this population number was seen (Citorik et al., 2014).

Antibiotic resistance is one of the growing issues globally. In order to precisely kill the strains from mix population, CRISPR has been developed that kills only pathogenic bacteria without killing any of the beneficial bacteria (Gomaa et al., 2014; Beisel et al., 2014; Bikard and Barrangou, 2017). Kim et al. (2016) used the CRISPR-Cas9 platform to target and kill the extended-spectrum beta-lactamase (ESBL)-producing E. coli. ESBL is commonly associated with Multi-drug resistance (MDR) and it is plasmid-mediated antibiotic resistance that can easily transfer horizontally into the bacterial community. Specifically targeting the antibiotic resistance gene re-sensitized the pathogen to its appropriate antibiotics. Park et al. (2017) used CRISPR-Cas9 antimicrobial for controlling S. aureus. They modified and improved efficacy and safety of CRISPR-Cas9 antimicrobial agent for therapeutic use in both in vivo and in vitro.

CRISPR-Cas9 system was employed for genome editing of Gram-positive bacteria Actinomycetales. Researchers have targeted two genes (actIORF1 and actVB) of the actinorhodin pathway of Streptomyces coelicolor A3(2) that led to its successful inactivation with 100% efficiency when templates for HDR were available (Tong et al., 2015). CRISPR-Cas9 system was used in yeast strain for the multiplex genome editing using different sgRNAs for targeting 5 different loci within genome such as bts1, yjI064w, erg9, ypl062W, rox1. The mutations in Saccharomyces cerevisiae were screened and found with 100 % efficiency. With this experiment, it was possible to knock-out a competitive pathway that ultimately increased the mevalonate production by 41-fold than the native strain (Jakočiūnas et al., 2015).

CRISPR-Cas9 system finds utility for controlling fungal infection in plants in order to increase crop productivity and yields. Amongst the many fungal pathogens, Phytophthora sojae is an oomycete (water mold) that infects the plant of agricultural and ornamental importance. CRISPR was used to target the Avr4/6 gene which belongs to the super-family of RXLR virulence effector proteins which could help to control fungal infection (Fang and Tyler, 2016). Codon-optimized Cas9 has shown improved ability for genome editing of a number of fungi including Aspergillus (Fuller et al., 2015; Katayama et al., 2016; Weber et al., 2017), β-lactam producing Penicillium chrysogenum (Pohl et al., 2016), Trichoderma reesei (higher cellulolytic enzyme producer) (Liu et al., 2015). CRISPR-Cas9 system can be explored more not only in humans, animals or microorganisms but also plants pathogens. As of now, the CRISPR-Cas9 system is currently less explored in plant-beneficiary fungi but most certainly it can be expanded to achieve better agricultural productivity and yields. CRISPR-Cas9 system has shown immense potential which should be expanded further toward biomedical, therapeutic, agricultural and industrial applications.

1.4.2. Viral genome editing using CRISPR-Cas9 system

Viruses are a small particles that can infect all organisms on the planet. Majority of viruses are known to cause animal and plant diseases. Currently, antiretroviral therapy (ART) is being used to control the viral infection but it cannot completely cure the disease. Sometimes, the viral genome may get integrated into the animal genome and latently reside there causing some serious diseases. CRISPR-Cas9 has revolutionized as an anti-viral therapy. It has been used to remove the viral infections in animals, plants (C chemokine receptor type 5 (CCR5) present on the white blood cells (WBCs) surface. CCR5 is an important receptor that facilitates the entry of HIV particle and it was observed, those people having a mutation in this gene (CCR5Δ32) are resistant to HIV infection. CRISPR-Cas9 has targeted the CCR5 gene and knocked-down its expression, conferring resistance against HIV infection (Wang et al., 2014; Li et al., 2015). Similarly, Ye et al. (2014) used CRISPR-Cas9 for targeting the CCR5 gene by deleting the 32nd-bp (CCR5Δ32). They modified iPSC and observed HIV resistance. There is another approach for HIV genome editing through CRISPR-Cas9, that is by targeting the long terminal repeat (LTR), wherein, the elimination of HIV allows to cure the infection (Ebina et al., 2013). Similarly, Hu et al. (2014) used single and multiplex sgRNA for the elimination of the HIV-1 genome by targeting the LTR U3 region which prevented the HIV infection.

Hepatitis B virus (HBV) is another serious human virus that causes liver infection (Zhang et al., 2014; Zeng, 2014). Lin et al. (2014) designed 8 sgRNAs for targeting the P1 and XCp of HBV and observed reduction of core HBsAg in Huh-7 hepatocytes. It was also tested in an animal model and complete clearance of HBV genome was seen. A reduction in the HBsAg serum level was observed. Many other reports are available where researchers used CRISPR-Cas9 for removal of HBV by targeting different region of genome (Zhen et al., 2015; Dong et al., 2015; Kennedy et al., 2015). CRISPR-Cas9 system can target genome of Epstein–Barr virus (EBV) in Raji (human cell line). It could arrest the proliferation and decrease the virus concentration (Wang and Quake, 2014). Similarly, two sgRNA along with Cas9 were used used for deleting a 558 bp sequence from promoter region of BART (BamHI A rightward transcript). It encodes viral microRNAs (miRNAs). It was found that loss of expression of those miRNAs were responsible for controlling of EBV infections (Yuen et al., 2015).

Human papillomavirus (HPV) is widely known to exhibit sexually transmitted infection worldwide and even causes cervical cancer. 100 different types of HPV are currently known, of which, 40 HPVs are passed through the sexual contact that can affect genital, mouth and throat (Schiffman et al., 2007). HPV E6 and E7 are major oncogenes which are responsible for progress of cancer. In a study, Kennedy et al. (2014) used CRISPR-Cas9 to target the E6 and E7 genes in HPV16 and control the infection. It was not very long ago that CRISPR-Cas9 based anti-viral agent was used to control wide range of viruses including Kaposi's sarcoma herpesvirus (KSHV) (Avey et al., 2015; van Diemen and Lebbink, 2017) and herpes simplex virus (HSV-1 and HSV-2) (Johnson et al., 2014; Diner et al., 2016; Xu et al., 2016; Wang et al., 2018). In conclusion, CRISPR-Cas9 could show high potential to control viral infection. CRISPR-Cas9 could be further extended for control and management of more viruses. Prior to the in vivo anti-viral therapy, a number of issues and challenges including delivery and off-target effect need to be addressed and tackled.

1.4.3. Mammalian cells genome editing using CRISPR-Cas9 system for therapeutic applications

In 2013, CRISPR-Cas9 system was implemented in the mammalian cells genome editing (Mali et al., 2013; Cong et al., 2013). Cong et al. (2013) developed a CRISPR-Cas9 system using the Cas9 gene from S. thermophilus, wherein, they designed and constructed a CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) to target mammalian genome using the 293FT cells and found efficient cleavage. Similarly, Mali et al. (2013) established CRISPR-Cas9 system for targeting endogenous AAVS1 locus where they engineered a chimeric crRNA-tracrRNA which is called as sgRNA. A number of mutations were found in 293T cells (10–25%), K562  cells (13-8%), and induced pluripotent stem cells (iPSCs) (2–4%). Both studies have opened up a new avenue and foundation for mammalian genome editing. In a study, Liang et al. (2015) developed liposome-mediated transfection/electroporation for Cas9/sgRNA ribonucleoprotein (RNP) complexes delivery into a number of mammalian cells. On targeting single loci in Jurkat T cells and iPSCs, 94% and 87% indels were found, respectively. Upon targeting multiple loci (2 or 3) the indel rate was 93% and 65%. There are a number of reports available to use CRISPR-Cas9 for therapeutic application for the prevention and treatment of animal diseases. Few of human diseases are given below which has been controlled using CRISPR-Cas9 technology.

1.4.3.1. Cancer therapy

The rapidly changing lifestyle, physical activity, foods and drugs have a positive and negative impact on our lives. High exposure to contamination leads to serious illness and health issue. Cancer is one of them and causes a high rate of mortality globally (Torre et al., 2015). In 2017, the US Food and Drug Administration approved a chimeric antigen receptor (CAR-T) immunotherapy. CRISPR-Cas9 was used to knock out a gene in T-cells that was linked with expression of CAR, which inactivates the programmed death-1 (PD-1) receptor in T-cells (Okada et al., 2017; Shao et al., 2017; Zhao et al., 2018). PD-1 is associated with regulation of anti-cancer cell immune response. The generated knock-out inhibited the receptor blocking ligands produced by cancer cells. Recently, numbers of studies have proven that targeting the PD-1 with CRISPR has notably increased the abundance of anti-tumor cytotoxic T-cells (in solid and hematologic cancers) (Shao et al., 2017; Zhao et al., 2018). These studies suggest that CRISPR-Cas9 has the potential to cure cancer and help for a better understanding of cancer biology.

1.4.3.2. Duchenne muscular dystrophy therapy

Duchenne muscular dystrophy (DMD) is a genetic disorder. It is characterized by progressive muscle degeneration and also weakness. Dystrophin is the protein responsible for causing DMD because of its gene expression. In order to create a model for DMD, a mutation in the dystrophin gene alters the gene function resulting in loss of muscle strength. More recently, CRISPR-Cas9 was used for repairing the mutation in dystrophin gene that allows to restore the gene function. The effectiveness and safety of CRISPR-Cas9 for DMD was checked in vitro and in vivo. It was tested and studied in a number of animals (Kim et al., 2017; Shimo et al., 2018; Koo et al., 2018; Maruyama and Yokota, 2018). In the future, more research needs to be done in order to properly treat DMD in human patients.

1.4.3.3. Beta-thalassemia therapy

β-Thalassemia is a blood disorder caused due to lack of hemoglobin production. A mutation in the globin gene leads to a deficiency of β-globin expression. Hemoglobin is an iron containing protein present in red blood cells (RBCs) and carries oxygen to cells of entire body. However, in the case of β-thalassemia, low expression of hemoglobin leads to less concentration of oxygen in many parts of body (Cao and Galanello, 2010). CRISPR-Cas9 was employed to repair the globin gene mutation and it was found to restore the function of globin for an adequate supply of oxygen into cells (Cyranoski and Reardon, 2015; Antony et al., 2018). Thus, it could be a potential solution to help the patient with β-thalassemia but it requires more experiments and in vivo studies to extend its reach to the clinic.

1.4.3.4. Blindness therapy

Blindness is a retinal degeneration disease globally. It is estimated that about 196 million people worldwide might develop this disease by 2020. The effective treatments for retinal degeneration by using drugs, gene therapy and transplantation have already been widely tried (Cai et al., 2018). Recently, CRISPR-Cas9 was applied to modify the retinitis pigmentosa which is the loss of cone photoreceptors that leads to blindness. This is one of the major therapeutic potentials to target treatment of blindness. Adeno-associated virus (AAV) -based CRISPR-Cas9 was developed for delivery of CRISPR-tool into post-mitotic photoreceptors that targets the Nrl gene (coding for neural retina-specific leucine zipper protein) which is rod fate determinant throughout the development of photoreceptor. It was found that the disruption of Nrl could be a promising solution for the treatment of blindness (Yu et al., 2017).

1.4.3.5. Cardiovascular disease therapy

Cardiovascular disease (CVD) is a heart-related disease and amongst the leading causes of death globally. Recently, CRISPR-Cas9 was employed for correction of a three-base-pair homozygous deletion in low-density lipoprotein cholesterol receptor (LDLR) exon 4 of iPSCs derived from a patient with familial hypercholesterolemia heterozygous (HoFH). It could normalize the cholesterol metabolism at the cellular level (Omer et al., 2017). G-protein–coupled estrogen receptor (Gper1) is associated with cardiovascular disease. In salt-sensitive hypertensive rats, deletion of Gper1 using CRISPR-Cas9 altered the microbiota, along with difference in the level of short chain fatty acids (SCFA) and improved the vascular relaxation. Waghulde et al. (2018) transplanted the microbiota from hypertensive Gper1+/+ rats. This reversed the cardiovascular protective effect exerted by deletion of Gper1. It suggests the role of Gper1 in accelerating the microbiota alterations which leads to cardiovascular disease. A wide range of uses of CRISPR-Cas9 for treatment of many serious animal diseases have been explored, still, a number of challenges and issues remain to be addressed before successful implementation into the clinic.

1.5. Recent developments in CRISPR interference platform

CRISPR interference (CRISPRi) has revolutionized gene repression, activation, high throughput screening, imaging, epigenetic modification and many more. This has been made possible by mutating the active region (RuvC (D10A) and HNH (H840A)) of Cas9 in order to attenuate the Cas9, yet retaining its binding ability. This modified version of the Cas9 protein is commonly called as dead Cas9 or dCas9 (CRISPRi) (Qi et al., 2013; Bikard et al., 2013; Chen et al., 2013; Gilbert et al., 2013; Ma et al., 2015) which has been extensively used number of organisms, model organisms and cell types.

1.5.1. CRISPRi

CRISPRi is repurposed for gene regulation in many organisms. The sgRNA is designed in a way to target the promoter region and coding gene that interfered the gene expression (Qi et al., 2013) and it has also been used for activating the endogenous gene in order to enhance the gene function (Bikard et al., 2013; Qi et al., 2013). It was initially tested in bacteria and mammalian cells and was eventually expanded in other organisms. In E. coli, sgRNA was used for blocking the transcription and 1000-fold repression with no off-target effects was found. Researchers could repress the sfGFP (superfolder green fluorescent protein) and mRFP (monomeric red fluorescent protein). In HEK293  cells (Human Embryonic Kidney 293  cells), eGFP (enhanced green fluorescent protein) was targeted which is expressed under control of SV40 promoter and showed up to 46% repression that has further improved by testing other sgRNA at different locations of gene (Qi et al., 2013). Similarly, Bikard et al. (2013) reported that CRISPRi interferes with RNA polymerase (RNAP) to bind with promoter sequences in order to block the transcription. They also targeted the transcriptional terminator using CRISPRi for blocking the running RNAP. GFP-mut2 was used to target the promoter key element and RBS, and a 100-fold repression was observed. A 20–40 folds and 6–35 folds repression were observed by targeting non-coding and coding strand, respectively. This study indicates that targeting non-coding is a better option than the coding region.

In order to increase the power of CRISPRi in eukaryotes, Gilbert et al. (2013) fused number of repressor domains with dCas9 and used them for gene repression. HEK293 cells chromosomally express GFP that was targeted by sgRNA. A 5-fold repression was observed when the cells expressed dCas9-KRAB fusion protein which was more than the previously reported (2-fold repression) (Qi et al., 2013). In another study, dCas9-KRAB fusion has been developed for silencing HS2 enhancer. It was found that the specific induction of H3K9 trimethylation (H3K9me3) leads to decrease in the chromatin accessibility (Thakore et al., 2015).

In order to repress many gene function together, a number of transcription factors are required. CRISPRi has the potential to insert cognate sequences and down-regulate range of genes together. CRISPRi has facilitated the building of transcriptional logic gates. NOT gate has been built by using 5 sigma 70 synthetic promoters that could repress up to 56–440 fold on-targets. Endogenously connected malT was targeted for changing the functionality of phage resistance, chemotaxis and sugar utilization (Nielsen and Voigt, 2014). CRISPRi has been applied in metabolic engineering for improving metabolites production. It has targeted the repression of pgi and pck gene in Corynebacterium glutamicum which allowed improving the L-lysine and L-glutamine production as compared to gene knock out of genes (Cleto et al., 2016).

1.5.2. CRISPRa

CRISPRi activates the gene function by fusing the activator domains with dCas9 (Bikard et al., 2013; Dominguez et al., 2016). The dCas9 was fused with ω-subunit RNA polymerase at N-and C-terminal region that targets the LacZ in E. coli. A 2.8-fold activation was found when dCas9-ω was present at C-terminal region. Bikard et al. (2013) have targeted the GFP-mut2 at different location and found 7.2–23 fold activation in gene expression.

In order to improve the mammalian gene activation, VP16 is well-known activator that is used for activating endogenous gene (Maeder et al., 2013; Gilbert et al., 2013; Perez-Pinera et al., 2013). In a study, dCas9 was fused with 4-copies of VP16 (VP64) and one copy of p65AD. It was transfected into a cell line (HEK293) to target Gal4 UAS (upstream activation sequence). 25-fold and 12-fold activation were observed when fusion protein contained activator dCas9-VP64 and dCas9-p65D, respectively (Gilbert et al., 2013). A number of reports support the use of CRISPRi for many functions in prokaryotes and eukaryotes. Currently, CRISPRi has the potential to easily regulate gene function in a simple way which is rapid, cost-effective and versatile.

1.5.3. Loci imaging

Tracking and visualization of genes on the chromosome for mapping and study are very important. CRISPRi is currently used for mapping of a gene on the chromosome and also visualization into cells. Chen et al. (2013) have developed a CRISPRi (fused dCas9-eGFP) for targeting the repetitive element present in telomere and coding genes for live imaging. They could identify and study the telomere dynamics during disruption and elongation. They also visualized the localization of MUC4 loci on sister chromatids and its dynamics. Anton et al. (2014) constructed CRISPRi by fusing of eGPF with dCas9 and targeted telomeric repeats, pericentric and centric. Similarly, Ma et al. (2015) engineered a multi-colour CRISPRi using the 3 different orthogonal dCas9 and targeted the many loci on chromosome. They could easily determine the distance between the two loci and physically map it. CRISPRi could be further expanded for mapping and visualization of a gene on the chromosome for a better understanding of the dynamics and regulation.

1.6. Conclusion and future remarks

CRISPR technology is rapidly expanding in many areas of genome editing, diagnostics, therapeutics, animal studies, biomedical and biotechnological applications, and industries. It has the potential to easily kill microbial pathogen and correct the gene mutation for curing of animal diseases (Singh et al., 2017, 2018). CRISPR-Cas9 designed to function as an antimicrobial agent can specifically kill harmful bacteria without hampering the beneficial bacteria (Bikard et al., 2014; Citorik et al., 2014). It has been integrated into the genome of bacteriophage for targeting of S. aureus which is one of the excellent delivery systems for removal of the pathogen. CRISPR-Cas9 has targeted many human viruses (HIV, HBV, and HPV). It has a great future for easily targeting and controlling viruses, not only from animals but also from plants in order to increase crop productivity. In the near future, CRISPR pill may be made available to use as a drug for controlling diseases.

We are suffering from a number of untreated genetic and heredity diseases. CRISPR-Cas9 technology is a key for repairing the gene correction in order to rescue the healthy condition. A number of serious human diseases are caused by a single gene mutation or defects in genes including cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, beta-thalassemia and many more. Editas Medicine (USA) has raised funding for CRISPR technology to treat these kinds of human diseases. More recently, CRISPR has been developed for rapid, specific and ultrasensitive detection and diagnosis of virus, cancer mutation and bacteria. This can be further used for precise and sensitive diagnosis for enabling proper treatment of diseases at an early stage.

CRISPRi currently plays a major role in gene regulation, loci imaging, high throughput screening, epigenetic modification and many more without altering the gene sequences. Off-target effects remain a challenging issue and further innovation in CRISPR can overcome many issues in order to use the full potential of technology in the coming year. Cas9 toxicity and its delivery is also a major concern and challenge that will be answered in future for realizing and reaching to the clinic for biomedical, industrial, therapeutic and biotechnological applications.

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