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Engineered CRISPR Systems for Next Generation Gene Therapies.
An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.
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Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.
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The Biology and Application Areas of CRISPR Technologies.
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Type II anti-CRISPR proteins as a new tool for synthetic biology.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.
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The better edge of the CRISPR blade.
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Expanding the Toolbox and Targets for Gene Editing.
Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.
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Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.
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Sharpening the CRISPR Toolbox.
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CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.
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CRISPR-Cas9 Probing of Infectious Diseases and Genetic Disorders.
The ability to precisely change the deoxyribonucleic acid (DNA) bases at specific sites offers tremendous advantages in the field of molecular biology and medical biotechnology. Identification of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), revelation of its role in prokaryotic adaptive immunity and subsequent conversion into genome and epigenome engineering system are the landmark research progresses of the decade. The possibilities of deciphering the molecular mechanisms of the disease, identifying the disease targets, generating the disease models, validating the drug targets, developing resistance to the infection and correcting the genotype have brought off much enthusiasm in the field of infectious diseases and genetic disorders. This review focuses on CRISPR/Cas9's impact in the field of infection and genetic disorders.
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