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CRISPR-Cas9 Structures and Mechanisms.
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
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Applications of CRISPR/Cas9 technology for modification of the plant genome.
The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to protect the cells from infection. This technology has now evolved to become an efficient genome editing tool, and is replacing older gene editing technologies. This technique uses programmable sgRNAs to guide the Cas9 endonuclease to the target DNA location. sgRNA is a vital component of the CRISPR technology, since without it the Cas nuclease cannot reach to its target location. Over the years, many tools have been developed for designing sgRNAs, the details of which have been extensively reviewed here. It has proven to be a promising tool in the field of genetic engineering and has successfully generated many plant varieties with better and desirable qualities. In the present review, we attempted to collect,collate and summarize information related to the development of CRISPR/Cas9 system as a tool and subsequently into a technique having a wide array of applications in the field of plant genome editing in attaining desirable traits like resistance to various diseases, nutritional enhancement etc. In addition, the probable future prospects and the various bio-safety concerns associated with CRISPR gene editing technology have been discussed in detail.
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Genome Editing of Rat.
Many genetically engineered rat strains have been produced for biomedical research. The simple and quick production of knock-out and knock-in rats is currently possible using genome editing techniques incorporating zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Genome-edited animals have been produced by the introduction of endonucleases into embryos using conventional microinjection and a new electroporation method named Technique for Animal Knockout system by Electroporation (TAKE). This chapter introduces the latest protocols for producing genetically engineered rats using genome editing.
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Highly efficient generation of biallelic reporter gene knock-in mice via CRISPR-mediated genome editing of ESCs.
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An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida).
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6-29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.
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CRISPR in personalized medicine: Industry perspectives in gene editing.
While research in gene editing technology has been active since the 1990s, recent advances in CRISPR ease of use have sparked significant enthusiasm in the biomedical community. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing has the potential to shape the therapeutic landscape of genetically-defined diseases, with key players such as CRISPR Therapeutics, Intellia, and Editas leading the way in β-thalassemia and sickle cell disease. In coming years, clinical development of CRISPR technology promises to change how we think about monogenic and polygenic diseases, though data is still in its infancy. While concerns regarding safety and administration of CRISPR persist and researchers are still seeking to better understand its effect on the body's ability to recognize and repair damaged DNA, the applications for personalized medicine are exciting and wide-ranging. This review examines potential applications of CRISPR for human therapeutics, with a private sector focus detailing anticipated clinical advances and evolving challenges along the way.
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CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose.
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Identifying drug-target selectivity of small-molecule CRM1/XPO1 inhibitors by CRISPR/Cas9 genome editing.
Validation of drug-target interaction is essential in drug discovery and development. The ultimate proof for drug-target validation requires the introduction of mutations that confer resistance in cells, an approach that is not straightforward in mammalian cells. Using CRISPR/Cas9 genome editing, we show that a homozygous genomic C528S mutation in the XPO1 gene confers cells with resistance to selinexor (KPT-330). Selinexor is an orally bioavailable inhibitor of exportin-1 (CRM1/XPO1) with potent anticancer activity and is currently under evaluation in human clinical trials. Mutant cells were resistant to the induction of cytotoxicity, apoptosis, cell cycle arrest, and inhibition of XPO1 function, including direct binding of the drug to XPO1. These results validate XPO1 as the prime target of selinexor in cells and identify the selectivity of this drug toward the cysteine 528 residue of XPO1. Our findings demonstrate that CRISPR/Cas9 genome editing enables drug-target validation and drug-target selectivity studies in cancer cells.
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CRISPR/Cas9 and cancer targets: future possibilities and present challenges.
All cancers have multiple mutations that can largely be grouped into certain classes depending on the function of the gene in which they lie and these include oncogenic changes that enhance cellular proliferation, loss of function of tumor suppressors that regulate cell growth potential and induction of metabolic enzymes that confer resistance to chemotherapeutic agents. Thus the ability to correct such mutations is an important goal in cancer treatment. Recent research has led to the developments of reagents which specifically target nucleotide sequences within the cellular genome and these have a huge potential for expanding our anticancer armamentarium. One such a reagent is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, a powerful, highly specific and adaptable tool that provides unparalleled control for editing the cellular genome. In this short review, we discuss the potential of CRISPR/Cas9 against human cancers and the current difficulties in translating this for novel therapeutic approaches.
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Targeted genome editing in primate embryos.
Mosaic mutations and off-target effects caused by CRISPR/Cas9 have led to concerns about the efficiency and specificity of this new technique in non-human primates and other large animals. Here we discuss recent findings from primate embryos, with a focus on the technical issues CRISPR/Cas9 faces before producing non-human primate models of human diseases.
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