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A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems.
Rapid, precise, and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications. Here, we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells. We construct the system by coupling Tet-on 3G and small molecule-assisted shutoff systems, which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules. This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression. The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence, allowing gene function study in a comprehensive expression profile. Our results reveal that the bidirectional control system enables sensitive dosage- and time-dependent regulation for either turn-on or shutoff of gene expression. We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats. The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.
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Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.
Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).
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CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.
BACKGROUND: The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. RESULTS: Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. CONCLUSIONS: The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.
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SCRAMBLER: A Tool for De Novo CRISPR Array Reconstruction and Its Application for Analysis of the Structure of Prokaryotic Populations.
CRISPR arrays are prokaryotic genomic loci consisting of repeat sequences alternating with unique spacers acquired from foreign nucleic acids. As one of the fastest-evolving parts of the genome, CRISPR arrays can be used to differentiate closely related prokaryotic lineages and track individual strains in prokaryotic communities. However, the assembly of full-length CRISPR arrays sequences remains a problem. Here, we developed SCRAMBLER, a tool that includes several pipelines for assembling CRISPR arrays from high-throughput short-read sequencing data. We assessed its performance with model data sets (Escherichia coli strains containing different CRISPR arrays and imitating prokaryotic communities of different complexities) and intestinal microbiomes of extant and extinct pachyderms. Evaluation of SCRAMBLER's performance using model data sets demonstrated its ability to assemble CRISPR arrays correctly from reads containing pairs of spacers, yielding a precision rate of >80% and a recall rate of 60-85% when checked against ground-truth data. Likewise, SCRAMBLER successfully assembled CRISPR arrays from the environmental samples, as attested by their matching with database entries. SCRAMBLER, an open-source software (github.com/biolab-tools/SCRAMBLER), can facilitate analysis of the composition and dynamics of CRISPR arrays in complex communities.
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CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia.
The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.).
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Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees.
In insects, odorant receptors facilitate olfactory communication and require the functionality of the highly conserved co-receptor gene orco. Genome editing studies in a few species of ants and moths have revealed that orco can also have a neurodevelopmental function, in addition to its canonical role in adult olfaction, discovered first in Drosophila melanogaster. To extend this analysis, we determined whether orco mutations also affect the development of the adult brain of the honey bee Apis mellifera, an important model system for social behavior and chemical communication. We used CRISPR/Cas9 to knock out orco and examined anatomical and molecular consequences. To increase efficiency, we coupled embryo microinjection with a laboratory egg collection and in vitro rearing system. This new workflow advances genomic engineering technologies in honey bees by overcoming restrictions associated with field studies. We used Sanger sequencing to quickly select individuals with complete orco knockout for neuroanatomical analyses and later validated and described the mutations with amplicon sequencing. Mutant bees had significantly fewer glomeruli, smaller total volume of all the glomeruli, and higher mean individual glomerulus volume in the antennal lobe compared to wild-type controls. RNA-Sequencing revealed that orco knockout also caused differential expression of hundreds of genes in the antenna, including genes related to neural development and genes encoding odorant receptors. The expression of other types of chemoreceptor genes was generally unaffected, reflecting specificity of CRISPR activity in this study. These results suggest that neurodevelopmental effects of orco are related to specific insect life histories.
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Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.
Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.
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Emerging therapies for type 1 diabetes: Immunotherapy and gene editing advances.
Type 1 diabetes is a chronic autoimmune disease involving the specific destruction of pancreatic β-cells and consequent absolute insulin deficiency. Although exogenous insulin replacement aids in hyperglycemic management, it does not cure the disease and increases the risks of hypoglycemia. Recent breakthroughs in disease-modifying therapies aim to delay, halt, or reverse disease progression via immune modulation or cell replacement. This narrative review systematically summarizes key therapeutic advances from 2023 to 2025. In immunotherapy, anti-CD3 monoclonal antibodies such as teplizumab have become the first Food and Drug Administration-approved agents to delay clinical onset in high-risk individuals. Inhibitors targeting pathways such as Janus kinase-STAT and engineered cell therapies such as chimeric antigen receptor-Tregs have also demonstrated potential for preserving residual β-cell function. For gene therapy and cell replacement, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing has generated "hypoimmune" stem cell-derived β-cells, with early clinical trials reporting instances of insulin independence without immunosuppression. Encapsulation technologies further provide a physical immune barrier for transplanted cells. Collectively, these advances mark a paradigm shift from symptomatic control to addressing core disease mechanisms through immune repair and functional restoration. Although challenges in long-term efficacy, safety, and accessibility persist, the ongoing development of combination strategies and precision medicine contributes to long-term remission and potentially a cure within reach.
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Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67phox-Deficient Chronic Granulomatous Disease.
Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67phox-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.
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Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.
The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.
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