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CRISPR-Based Therapies: Revolutionizing Drug Development and Precision Medicine.
With the discovery of CRISPR-Cas9, drug development and precision medicine have undergone a major change. This review article looks at the new ways that CRISPR-based therapies are being used and how they are changing the way medicine is done. CRISPR technology's ability to precisely and flexibly edit genes has opened up new ways to find, validate, and develop drug targets. Also, it has made way for personalized gene therapies, precise gene editing, and advanced screening techniques, all of which hold great promise for treating a wide range of diseases. In this article, we look at the latest research and clinical trials that show how CRISPR could be used to treat genetic diseases, cancer, infectious diseases, and other hard-to-treat conditions. However, ethical issues and problems with regulations are also discussed in relation to CRISPR-based therapies, which shows how important it is to use them safely and responsibly. As CRISPR continues to change how drugs are made and used, this review shines a light on the amazing things that have been done and what the future might hold in this rapidly changing field.
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Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS.
RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, also provide powerful tools to ablate specific genes. Because of differences in co-receptor usage and the high mutability of the HIV-1 genome, a combination of host factors and viral genes needs to be suppressed for effective prevention and treatment of HIV-1 infection. Whereas the continued presence of small interfering/short hairpin RNA (si/shRNA) mediators is needed for RNAi to be effective, the continued expression of nucleases in the gene-editing systems is undesirable. Thus, RNAi provides the only practical way for expression of multiple silencers in infected and uninfected cells, which is needed for effective prevention/treatment of infection. There have been several advances in the RNAi field in terms of si/shRNA design, targeted delivery to HIV-1 susceptible cells, and testing for efficacy in preclinical humanized mouse models. Here, we comprehensively review the latest advances in RNAi technology towards prevention and treatment of HIV-1.
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Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.
The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.
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Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.
γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1-5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.
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MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries.
SUMMARY: The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9-12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes. AVAILABILITY AND IMPLEMENTATION: MULTIGUIDESCAN IS AVAILABLE AT GITHUB: https://github.com/bioinfomaticsCSU/MultiGuideScan. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.
Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
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CRISPR innovations in tissue engineering and gene editing.
The CRISPR/Cas9 system is a powerful tool for genome editing, utilizing the Cas9 nuclease and programmable single guide RNA (sgRNA). However, the Cas9 nuclease activity can be disabled by mutation, resulting in catalytically deactivated Cas9 (dCas9). By combining the customizable sgRNA with dCas9, researchers can inhibit specific gene expression (CRISPR interference, CRISPRi) or activate the expression of a target gene (CRISPR activation, CRISPRa). In this review, we present the principles and recent advancements of these CRISPR technologies, as well as their delivery vectors. We also explore their applications in stem cell engineering and regenerative medicine, with a focus on in vitro stem cell fate manipulation and in vivo treatments. These include the prevention of retinal and muscular degeneration, neural regeneration, bone regeneration, cartilage tissue engineering, and the treatment of blood, skin, and liver diseases. Furthermore, we discuss the challenges of translating CRISPR technologies into regenerative medicine and provide future perspectives. Overall, this review highlights the potential of CRISPR in advancing regenerative medicine and offers insights into its application in various areas of research and therapy.
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Controlling and enhancing CRISPR systems.
Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature.
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The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.
Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.
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Reading and writing: the evolution of molecular pain genetics.
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