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A programmable sequence of reporters for lineage analysis.
We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.
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Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management.
The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.
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Efficient gene editing of a model fern species through gametophyte-based transformation.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P < 0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications.
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Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens.
Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.
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Stomatal XVE: an inducible system for cell-stage-specific gene expression and editing in the stomatal lineage.
Stomatal development has emerged as a valuable model for studying developmental processes. Examining gene function along the stomatal lineage often requires gene perturbation in a controlled and cell-stage-specific manner, but this remains tedious without a dedicated genetic tool. Here, we describe Stomatal XVE, a modular, two-component XVE-based inducible system that enables user-controlled gene overexpression and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based knockout at defined stomatal cell stages in Arabidopsis thaliana. The system consists of a collection of estrogen-responsive XVE driver lines under cell-stage-specific promoters and effector vectors responsive to activated XVE. This design simplifies cloning and allows users to scale their investigation. We validated the cell-stage specificity and inducibility of the XVE driver lines and characterized key induction parameters. To test the system functionally, we employed it to study MAPKKK YODA and a pathogen effector AvrPtoB. While YODA overexpression reproduced known early- and late-stage phenotypes, stage-specific knockouts argued against its late-stage role in guard cell (GC) differentiation. Furthermore, AvrPtoB expression during later stages triggered striking disruptions in GC morphology and viability, revealing cell-type-specific effects of the pathogen protein. Overall, our Stomatal XVE system enables precise functional analysis of genes across defined stages of stomatal development and is particularly well suited for investigating genes with pleiotropic effects.
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Diazaquinomycin Biosynthetic Gene Clusters from Marine and Freshwater Actinomycetes.
Tuberculosis is an infectious disease of global concern. Members of the diazaquinomycin (DAQ) class of natural products have shown potent and selective activity against drug-resistant Mycobacterium tuberculosis. However, poor solubility has prevented further development of this compound class. Understanding DAQ biosynthesis may provide a viable route for the generation of derivatives with improved properties. We have sequenced the genomes of two actinomycete bacteria that produce distinct DAQ derivatives. While software tools for automated biosynthetic gene cluster (BGC) prediction failed to detect DAQ BGCs, comparative genomics using MAUVE alignment led to the identification of putative BGCs in the marine Streptomyces sp. F001 and in the freshwater Micromonospora sp. B006. Deletion of the identified daq BGC in strain B006 using CRISPR-Cas9 genome editing abolished DAQ production, providing experimental evidence for BGC assignment. A complete model for DAQ biosynthesis is proposed based on the genes identified. Insufficient knowledge of natural product biosynthesis is one of the major challenges of productive genome mining approaches. The results reported here fill a gap in knowledge regarding the genetic basis for the biosynthesis of DAQ antibiotics. Moreover, identification of the daq BGC shall enable future generations of improved derivatives using biosynthetic methods.
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A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.
CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9's activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.
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Hypoimmunogenic Human Pluripotent Stem Cells as a Powerful Tool for Liver Regenerative Medicine.
Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the β2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.
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Platinum TALEN-mediated nonviral gene editing facilitates clinical-scale production of cancer antigen-reactive T cells.
BACKGROUND AIMS: Recently, target-genome editing has emerged as a next-generation tool for the clinical development of designed cellular products. Although Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) is the most frequently adopted nuclease for therapeutic genome editing, its widespread use is still hampered by potential off-target effects and high patent royalties. Platinum transcription activator-like effector nuclease (TALEN) is a modified TALEN that harbors non-repeat-variable di-residue (non-RVD) variations and confers higher efficiency than conventional TALENs lacking non-RVD variations. METHODS: In this study, using Platinum TALEN targeting T-cell receptor (TCR) gene loci and a single-stranded DNA homology-directed repair (HDR) template, we aimed to produce TCR-replaced human T cells reprogrammed to recognize a cancer antigen and kill cancer cells. RESULTS: This system can reproducibly produce TCR-engineered T cells from ∼50 mL of peripheral blood on a clinical scale. The resulting genome-edited T cells retain naïve/naïve-like and memory phenotype cells in both CD4+ and CD8+ fractions and exhibit efficient cytolytic activity against cancer cell lines in vitro. CONCLUSION: In conclusion, Platinum TALEN-mediated nonviral genome editing facilitates the replacement of the endogenous TCR with a desired TCR and can be applied to the clinical manufacturing of therapeutic T-cell products.
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Cell-penetrating peptides and their utility in genome function modifications (Review).
For almost 30 years, studies have confirmed the effectiveness of cell-penetrating peptides (CPPs) in the facilitation of the intracellular delivery of various cargo molecules, including RNA, DNA, plasmids, proteins or nanoparticles, under in vitro and in vivo conditions. The cellular uptake of CPPs occurs via energy-dependent, as well as -independent mechanisms. In this relatively new direction of research, studies have attempted to introduce genome modification systems into cells by CPPs. Cellular uptake of CPPs carrying either covalently bound or electrostatically conjugated cargo, has several advantages over viral delivery systems, as it does not lead to any significant cytotoxicity or immunogenicity, and simultaneously it is more efficient than other non-viral systems. So far, CPPs have been successfully used to introduce Cre recombinase, zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats systems into cells. The present article systematically reviewed the information obtained from studies on CPPs and assessed their utility with regard to their effectiveness and safety of use.
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