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Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences.
CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.
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CRISPR technology for diagnosis and treatment of human brucellosis.
The global burden of Brucella infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of Brucella in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the Brucella replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant Brucella strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global Brucella infections.
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From hacking the human genome to editing organs.
In the recent decades, human genome engineering has been one of the major interesting research subjects, essentially because it raises new possibilities for personalized medicine and biotechnologies. With the development of engineered nucleases such as the Zinc Finger Nucleases (ZFNs), the Transcription activator-like effector nucleases (TALENs) and more recently the Clustered Regularly Interspaced short Palindromic Repeats (CRISPR), the field of human genome edition has evolved very rapidly. Every new genetic tool is broadening the scope of applications on human tissues, even before we can completely master each of these tools. In this review, we will present the recent advances regarding human genome edition tools, we will discuss the numerous implications they have in research and medicine, and we will mention the limits and concerns about such technologies.
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From CRISPR scissors to virus sensors.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas Advancement in Molecular Diagnostics and Signal Readout Approaches.
Discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has greatly improved the gene editing technology. Their applications in the area of diagnostic innovation are gaining much attention. The key characteristics of CRISPR/Cas system that allowed its extensive exploitation in the detection platform are their programmable and highly selective target recognition scheme. Herein, this review presents the major three Cas effectors (Cas9, Cas12, and Cas13) and their significance in various detection assays. The CRISPR/Cas detection strategies, based on their target hybridization, cleavage activity, sensor capabilities, and signal readout methods, are discussed. Some of the recent progressions, challenges, and improvement strategies of CRISPR/Cas technology are highlighted and their biosensing detection platforms toward the development of simple, sensitive, and portable point-of-care diagnostic devices are presented.
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CRISPR screens beyond Cas9.
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Type III CRISPR-Cas System: Introduction And Its Application for Genetic Manipulations.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes provide adaptive immunity against invasion of foreign nucleic acids in archaea and bacteria. The system functions in three distinct stages: adaptation, biogenesis, and interference. CRISPR-Cas systems are currently classified into at least five different types, each with a signature protein among which Type III systems exhibit a dual DNA/RNA interference activity. Structures of a few Type III surveillance complexes have been determined: they are composed of several different subunits and exhibit striking architectural similarities to Type I surveillance complexes. Here, we review the genetic, biochemical, and structural studies concerning CRISPR-Cas Type III systems and discuss their application for genetic manipulations, including genome engineering and gene silencing.
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Five Years of Progress in CRISPR Clinical Trials (2019-2024).
In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both in vivo and ex vivo gene and cell therapies.
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From genome scissors to molecular scalpel: evolution of CRISPR systems.
From bizarre palindromic repeats to a bacterial defense mechanism, to genome editing tool, and more, Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR has significantly impacted the way we study genome modification in less than a decade. In this review, we would like to highlight some key players over 30 years of research and explain this biotechnological tool's basic mechanisms. We also refer to the evolution of the CRISPR variants and some of the applications derived from them. The understanding and upgrading of this system will be a valuable tool in the years to come to solve some of the challenges in diverse fields from pharmaceuticals to therapeutics, from basic plant genetics to crop improvement, from metabolic engineering to waste management and industrial processing.
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Second installment of Special Issue on expanding the scale and scope and of gene editing.
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