CRISPR continues to drive the world of gene editing. About seven years ago, scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells. Since then, interest in the method has spread extensively across the globe. Not long after its sudden headstart in 2013, already thousands of laboratories started taking up the technique and investors began funding startups to harness its potential. This ultimately resulted in major improvements being made in incredibly short periods of time. Simultaneously, this also initiated and continues to drive an increasing gap between new innovative applications in the field of gene editing and the overall awareness of the general public. Ethical concerns have remained a steady companion of this rise. With myriads of research papers about CRISPR related topics being published every year, trying to stay on top of developments can be a cumbersome task.
The goal of this paper is to summarize current practice and research areas that are part of modern gene editing. Following a brief summary of CRISPR basics, used endonucleases and techniques, its impact on the treatment of hereditary diseases, viral infections and cancer is illustrated based on recent examples. Effects of gene editing on cell line engineering efforts are described using studies focusing on glycosylation, impurities, cultivation and production efficiency issues of CHO cell lines. Besides discussing CRISPR applications for rapid SARS-CoV-2 diagnosis, current challenges for clinical use including off-targets, immune responses and lacking drug delivery efficiencies as well as promising developments are being reviewed. This paper is supposed to support quick elucidation of future potential of the rapidly evolving world of CRISPR Cas genome editing and facilitate retrieval of further literature.
Contents
1 Introduction
2 State-of-the-art Gene Editing
2.1 PAM sequence
2.2 Type II CRISPR-Cas System - Cas9 variants and orthologs
2.2.1 dCas9
2.2.2 eSpCas9
2.2.3 SpCas9-HF1
2.2.4 HypaCas
2.2.5 xCas
2.2.6 Cas9 Nickases
2.2.7 Remarkable SpCas9 Orthologs
2.2.8 Base editing
2.2.9 Prime editing
2.3 Type V CRISPR-Cas System - Diagnostic tools
2.3.1 Cas12a, Cas12b
2.4 Type VI CRISPR-Cas System - RNA targeting
2.4.1 Cas13a
2.4.2 CasRx
2.5 Transfection methods
2.5.1 Non-viral
2.5.2 Viral
3 Gene Editing and Treatment of Diseases
3.1 Hemoglobinopathies
3.1.1 CRISPR-correction of sickle cell disease
3.1.2 Reactivation of Fetal Hemoglobin Production
3.2 Cystic fibrosis
3.2.1 Functional repair of the CFTR gene
3.2.2 CFTR repair using Adenine Base Editors
3.3 Duchenne muscular dystrophy (DMD)
3.3.1 CRISPR-Cas9 restores functional dystrophin in mice
3.3.2 Long-term evaluation of Dystrophin restoration in mice
3.4 Treatment of Human Immunodeficiency Virus
3.4.1 Genome editing of HIV co-receptors CCR5 and CXCR4
3.4.2 HIV elimination using ART and CRISPR-Cas9
3.5 Cancer immunotherapy
3.5.1 Editing Primary Human Natural Killer Cells
3.5.2 Creating enhanced CAR T Cells using CRISPR-Cas9
3.5.3 Creating universal CAR T cells with CRISPR-Cas9
4 Gene Editing for Drug Development
4.1 Glycoengineering
4.2 Prolonged cultivation
4.3 Elimination of host cell protein impurities
4.4 Rapid adaptation cells
4.5 Enhanced protein production
5 Gene Editing Tools for Diagnostics
5.1 Diagnostic CRISPR endonucleases
5.1.1 Cas12a, Cas12b
5.1.2 Advanced endonucleases for diagnostic
5.1.3.. Cas13a
5.2 SHERLOCK
5.2.1 SARS-CoV-2 Detection using SHERLOCK
5.3 DETECTR 29
5.3.1 SARS-CoV-2 Detection using DETECTR
5.4 CRISPR targeting of drug-resistant bacteria
5.4.1 Targeting pathogenic and drug-resistant bacteria
6 Challenges and Obstacles
6.1 Off-target effects and carcinogenesis
6.2 Delivery of the CRISPR-Cas system
6.3 Immune responses against CRISPR-Cas
7 Prospect
List of Figures
List of Abbreviations
Bibliography
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