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Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens.
We mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome primarily responsible for inhibiting gravitropism, proving the utility of the mutant library. The CRISPR-Cas9 system is a powerful tool for genome editing. Here we report highly-efficient multiplex CRISPR-Cas9 editing of the seven-member phytochrome gene family in the model bryophyte Physcomitrium (Physcomitrella) patens. Based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to identify high-order multiple mutants prior to sequencing, we demonstrate successful targeting of all seven PHY genes in a single transfection. We investigated further aspects of the CRISPR methodology in Physcomitrella, including the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repairing the chromosome when excising a complete locus. As proof-of-principle, we show that the septuple phy- mutant remains gravitropic in light, in line with expectations, and on the basis of data from lower order multiplex knockouts conclude that phy5a is the principal phytochrome responsible for inhibiting gravitropism in light. We expect, therefore, that this mutant collection will be valuable for further studies of phytochrome function and that the methods we describe will allow similar approaches to revealing specific functions in other gene families.
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Short cell-penetration peptide conjugated bioreducible polymer enhances gene editing of CRISPR system.
CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.
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Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.
CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.
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Gene Therapy for Alzheimer and Parkinson Diseases.
Alzheimer and Parkinson diseases are associated with cholinergic neuron loss and deterioration of bone mineral density. Gene therapy through either gene transfer, CRISPR gene editing, or CRISPR gene modulation holds the potential to cure Alzheimer and Parkinson diseases. The emerging role of weight-bearing exercise in the prevention of, and care for, osteoporosis, obesity, and diabetes has been previously recognized. Moreover, endurance exercise offers a viable alternative to reduce amyloid peptides deposits while increasing bone mineral density in Alzheimer and Parkinson patients. β-amyloid peptides, α-synuclein, and tau aggregates start building up two decades before the onset of Alzheimer and Parkinson diseases. Therefore, an early intervention program for the detection of these deposits is required to prevent or delay the onset of these diseases. This article spots light on the potential of gene therapy for Alzheimer and Parkinson diseases.
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High-throughput CRISPR technology: a novel horizon for solid organ transplantation.
Organ transplantation is the gold standard therapy for end-stage organ failure. However, the shortage of available grafts and long-term graft dysfunction remain the primary barriers to organ transplantation. Exploring approaches to solve these issues is urgent, and CRISPR/Cas9-based transcriptome editing provides one potential solution. Furthermore, combining CRISPR/Cas9-based gene editing with an ex vivo organ perfusion system would enable pre-implantation transcriptome editing of grafts. How to determine effective intervention targets becomes a new problem. Fortunately, the advent of high-throughput CRISPR screening has dramatically accelerated the effective targets. This review summarizes the current advancements, utilization, and workflow of CRISPR screening in various immune and non-immune cells. It also discusses the ongoing applications of CRISPR/Cas-based gene editing in transplantation and the prospective applications of CRISPR screening in solid organ transplantation.
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A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo.
DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.
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Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors.
CRISPR (clustered regularly interspaced short palindromic repeat) system-based precise genome editing remarkably impacts both scientific investigation and therapeutic practices. Among various techniques, DNA donor-mediated homology-directed repair (HDR) represents a promising method for precise gene editing. Although efficiency constraints have previously limited HDR, recent advancements have significantly enhanced its effectiveness. Therefore, it is essential to highlight the progress made in this field and to reassess the potential of the HDR approach. In this review, we explore the fundamental principles of HDR-dependent gene editing and evaluate current strategies to enhance HDR efficiency, with particular emphasis on single-stranded DNA (ssDNA) donor-mediated HDR. Finally, we discuss the prospects of high-efficiency ssDNA donor-mediated precise gene editing in laboratory research and clinical therapies.
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CRISPR/Cas9-Enabled Multiplex Genome Editing and Its Application.
The CRISPR/Cas9 system is a prevalent and versatile genome-editing tool of choice for basic and applied biological research. An exchange of a 20-bp spacer sequence in the gRNA can easily reprogram Cas9 to target a different DNA site. By expressing or providing multiple gRNAs, the system also enables multiplex genome editing at high efficiencies. Current approaches for providing multiple gRNAs in vivo include the use of multigene cassettes to express several gRNAs, Csy4-based excision, arrays of crRNAs, ribozyme-flanked gRNAs, tRNA-dependent cleavage of gRNAs, and direct introduction of Cas9 proteins preloaded with different gRNAs. By simultaneously targeting multiple DNA sequences, multiplex genome editing can be used to knockout multiple genes or delete chromosomal fragments. Off-target risk can also be reduced by Cas9-dimers that require the simultaneous expression of two gRNAs. With multiple gRNAs, specific gene expression or methylation status can be efficiently controlled by dCas9 fused to activators, repressors, methyltransferase, demethylase, or other functional domains. As a result, multiplex genome editing is expected to accelerate functional discovery of plant genes as well as genetic improvement of agricultural crops.
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TopCas: Topology-Gated Cas12a via DNA-RNA Chimeric Circular crRNA for Amplification-Free Nucleic Acid Detection and Conditional Gene Editing.
Controlled activation of CRISPR-Cas12a is critical for achieving conditional gene editing and molecular diagnostics. As an indispensable component for forming an active complex, CRISPR RNA (crRNA) represents a key route to regulate Cas12a activity. Here, we establish TopCas (Topology-gated Cas12a via DNA-RNA Chimeric Circular crRNA) as a platform for preamplification-free nucleic acid detection and conditional gene editing. Within TopCas, the circular crRNA sterically constrains Cas12a's nuclease activity until target-activated complexes trans-cleave the DNA segment of the chimeric crRNA, converting the circular guide into its linear form and initiating an autocatalytic cascade that culminates in fluorophore release and signal amplification. By the same mechanism, the system conditionally activates Cas12a's gene-editing function (cis-cleavage) exclusively in the presence of specific nucleic acid targets (e.g., viral DNA or RNA). We demonstrate that TopCas affords high specificity and sensitivity in nucleic acid detection, supports accurate detection in clinical viral nucleic acid samples, and shows potential for in vivo real-time molecular imaging, while also demonstrating the feasibility of conditional gene editing. This innovative chimeric circular crRNA-Cas12a system not only provides a new tool for precise disease diagnostics but also offers a promising strategy for personalized therapeutic intervention.
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Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.
Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.
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