Previous Editing Research Articles

CRISPR-Cas9: A Cutting-edge Genome-editing Technology with Many Potential Applications

Unregulated human activities are responsible for climate change which is a major factor for the emergence of new infectious (bacterial, viral, fungus, and parasitic) and non-infectious diseases (genetic disorders, cancers, etc.). To tackle this situation, we must have better therapeutics, diagnostics, and vaccines for the treatment and prevention of emerging diseases in humans and animals. To address the aforementioned issues, CRISPR-Cas9, a revolutionary genome editing tool, can help us develop better therapeutics, diagnostics, and vaccines in a short time. In addition, it can be used to achieve food security by improving productivity, adaptability, and resilience traits in plants and animals. In nature, most bacteria and archaea have CRISPR as a part of their adaptive immune system that helps bacteria defend themselves from phages, viruses, and other foreign genetic elements. CRISPR-Cas9 is a highly effective, simple, and accurate genome editing tool compared to previous genome editing tools such as meganucleases, Zinc finger nucleases (ZFNs), and transcription activators like effector nucleases (TALENs). This review article discusses the different components and working of the CRISPR-Cas9 system, and how CRISPR-Cas9 plays a significant role in the development of next-generation therapeutics, diagnostics, vaccine platforms, and improved crops and livestock species for food security.

Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling.

Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light-oxygen-voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.

Image-to-Image Translation with Disentangled Latent Vectors for Face Editing.

We propose an image-to-image translation framework for facial attribute editing with disentangled interpretable latent directions. Facial attribute editing task faces the challenges of targeted attribute editing with controllable strength and disentanglement in the representations of attributes to preserve the other attributes during edits. For this goal, inspired by the latent space factorization works of fixed pretrained GANs, we design the attribute editing by latent space factorization, and for each attribute, we learn a linear direction that is orthogonal to the others. We train these directions with orthogonality constraints and disentanglement losses. To project images to semantically organized latent spaces, we set an encoder-decoder architecture with attention-based skip connections. We extensively compare with previous image translation algorithms and editing with pretrained GAN works. Our extensive experiments show that our method significantly improves over the state-of-the-arts. Project page: https://yusufdalva.github.io/vecgan.

PFNet: Attribute-aware personalized fashion editing with explainable fashion compatibility analysis

Attribute-aware editing provides a feasible way for users to participate in fashion design. In a sense, an explainable model of fashion compatibility can assist users to perceive the fashionability of their design. However, previous fashion editing researches are opaque in compatibility and rely on labels to manipulate the editing of specific coarse-grained attributes. Consequently, we propose a novel attribute-aware personalized fashion editing network with explainable fashion compatibility modeling, named PFNet, which can simultaneously decouple entangled attributes to make them editable and generate an attribute-wise compatibility explanation for fashion design. In PFNet, we propose an unsupervised garment attribute decoupling network, which independently encodes attributes by hierarchical style control and minimizing mutual correlation. Besides, we develop an attribute compatibility-aware attention network to deeply explore compatible interactions of attributes and visualize their internal decisions. The empirical experiments and user study on the FashionVC and Polyvore datasets reveal that the decoupling accuracy of PFNet for multiple clothing attributes is increased by an average of 22% compared to the state-of-the-art method, and provides more popular compatibility insights with an accuracy rate of 75.5%.

Nme2 Cas9-mediated therapeutic editing in inhibiting angiogenesis after wet age-related macular degeneration onset.

Age-related macular degeneration (AMD), particularly wet AMD characterised by choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway contributes to CNV pathogenesis. Previous gene editing research indicated that disrupting these genes in retinal pigment epithelial cells could have a preventive effect on CNV progression. However, no studies have yet been conducted using gene editing to disrupt VEGF signalling after CNV induction for therapeutic validation, which is critical to the clinical application of wet AMD gene editing therapies. Here, we employed the single-adeno-associated virus-mediated Nme2 Cas9 to disrupt key molecules in VEGF signalling, Hif1α, Vegfa and Vegfr2 after inducing CNV and estimated their therapeutic effects. We found that Nme2 Cas9 made efficient editing in target genes up to 71.8% post 11 days in vivo. And only Nme2 Cas9-Vegfa treatment during the early stage of CNV development reduced the CNV lesion area by 49.5%, compared to the negative control, while Nme2 Cas9-Hif1α or Nme2 Cas9-Vegfr2 treatment did not show therapeutic effect. Besides, no off-target effects were observed in Nme2 Cas9-mediated gene editing in vivo. This study provides proof-of-concept possibility of employing Nme2 Cas9 for potential anti-angiogenesis therapy in wet AMD.

CRISPR-engineered microalgae: a promising approach for the production of therapeutic proteins

Clustered regularly interspaced short palindromic repeats (CRISPR) has emerged as a transformative tool in biotechnology, providing precision in DNA modification through guide RNA (gRNA)-directed Cas9 nuclease. It surpasses previous genome editing techniques due to its simplicity in gRNA design and the ability to target multiple genes concurrently. This review explores the profound impact of CRISPR on research and clinical applications, particularly in the field of therapeutic protein production. Currently, therapeutic protein production relies on complex mammalian cell culture systems, burdened by limitations and contamination risks. In this context, microalgae provide a promising alternative. These unicellular organisms possess exceptional growth rates, enabling rapid and high-volume production of valuable products while thriving in diverse aquatic environments. They utilize renewable resources like sunlight and carbon dioxide, aligning with eco-friendly production principles. Microalgae's capacity to yield substantial quantities of proteins, lipids, and carbohydrates makes them an economically attractive and ecologically responsible platform for various industries. This review presents a comprehensive overview of recent advancements and challenges in CRISPR-engineered microalgae for therapeutic protein production. It discusses the advantages and drawbacks of various microalgal species, CRISPR tools, and delivery methods, as well as protein expression. Furthermore, it highlights the potential applications and advantages of microalgal biopharmaceuticals and offers insights into future directions to enhance the efficiency, safety, and scalability of CRISPR-based microalgal biotechnology.

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva.

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

Role of CRISPR-Cas9 in agricultural science

Clustered regularly interspaced short palindromic repeat (CRISPR), a potent gene-editing tool was found in 2012. CRISPR is a genetic engineering technique that enables genome editing in living creatures and is based on the bacterial CRISPR-Cas9 antiviral defense mechanism. It is simpler, less expensive, and more accurate than previous gene editing techniques. It also has a wide range of valuable uses, including improving crops and treating genetic diseases. Plant science has benefited more from the CRISPR/Cas9 editing technique than medical science. CRISPR/Cas9 has been used in a range of crop-related research and development domains, including disease resistance, plant development, abiotic tolerance, morphological development, secondary metabolism, and fiber creation, as a well-developed cutting-edge biotechnology technique. This paper summarized the role of the CRISPR-CAS9 tool in modern agricultural science.

Visual function restoration in a mouse model of Leber congenital amaurosis via therapeutic base editing

Leber congenital amaurosis (LCA), an inherited retinal degeneration, causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants, such as RPE65, are evident in specific genes, related to the functions of retinal pigment epithelium and photoreceptors. In contrast to the original Cas9, base editing tools can correct pathogenic substitutions without generation of DNA double-stranded breaks (DSBs). In this study, dual adeno-associated virus (AAV) vectors containing split adenine base editors (ABEs) with trans-splicing intein were prepared for invivo base editing in retinal degeneration of 12 (rd12) mice, an animal model of LCA, possessing a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). Subretinal injection of AAV-ABE in retinal pigment epithelial cells of rd12 mice resulted in an A to G transition. The on-target editing was sufficient for recovery of wild-type mRNA, RPE65 protein, and light-induced electrical responses from the retina. Compared with our previous therapeutic editing strategies using Cas9 and prime editing, or with the gene transfer strategy shown in the current study, our results suggest that, considering the editing efficacy and functional recovery, ABEs could be a strong, reliable method for correction of pathogenic variants in the treatment of LCA.

Recent Progress and Future Prospect of CRISPR/Cas-Derived Transcription Activation (CRISPRa) System in Plants.

Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed.

CRISPR-Cas9: Taming protozoan parasites with bacterial scissor.

The invention of CRISPR-Cas9 technology has opened a new era in which genome manipulation has become precise, faster, cheap and more accurate than previous genome editing strategies. Despite the intricacies of the genomes associated with several protozoan parasites, CRISPR-Cas9 has made a substantial contribution to parasitology. The study of functional genomics through CRISPR-Cas9 mediated gene knockout, insertion, deletion and mutation has helped in understanding intrinsic parasite biology. The invention of CRISPR-dCas9 has helped in the programmable control of protozoan gene expression and epigenetic engineering. CRISPR and CRISPR-based alternatives will continue to thrive and may aid in the development of novel anti-protozoan strategies to tame the protozoan parasites in the imminent future.

The CRISPR Revolution in Genome Engineering: Perspectives from Religious Ethics

ABSTRACTThis focus issue considers the normative implications of the recent emergence in genome editing technology known as CRISPR (clustered regularly interspaced short palindromic repeats) or CRISPR‐associated protein 9. Originally discovered in the adaptive immune systems of bacteria and archaea, CRISPR enables researchers to make efficient and site‐specific modifications to the genomes of cells and organisms. More accessible, precise, and economic than previous gene editing technologies, CRISPR holds the promise of not only transforming the fields of genetics, agriculture, and human medicine, but also heralding a new era of democratized biotechnology. However, the speed with which developments in the field have progressed threatens to overwhelm our normative sensibilities about the long‐term practical and ethical implications. The contributors to this focus issue attempt to think through some of the more salient moral and practical consequences of CRISPR in the context of religious ethics, particularly as they relate to themes of autonomy, human flourishing, social justice, and the ethics of enhancement.

Lamellipodia-like actin networks in cells lacking WAVE regulatory complex.

ABSTRACTCell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.

A new gain-of-function OsGS2/GRF4 allele generated by CRISPR/Cas9 genome editing increases rice grain size and yield

Grain size is one of the most important factors affecting rice grain quality and yield, and attracts great attention from molecular biologists and breeders. In this study, we engineered a CRISPR/Cas9 system targeting the miR396 recognition site of the rice GS2 gene, which encodes growth-regulating factor 4 (OsGRF4) and regulates multiple agronomic traits including grain size, grain quality, nitrogen use efficiency, abiotic stress response, and seed shattering. In contrast to most previous genome editing efforts in which indel mutations were chosen to obtain null mutants, a mutant named GS2E carrying an in-frame 6-bp deletion and 1-bp substitution within the miR396-targeted sequence was identified. GS2E plants showed increased expression of GS2 in consistent with impaired repression by miR396. As expected, the gain-of-function GS2E mutant exhibited multiple beneficial traits including increased grain size and yield and bigger grain length/width ratio. Thousand grain weight and grain yield per plant of GS2E plants were increased by 23.5% and 10.4%, respectively. These improved traits were passed to hybrids in a semi-dominant way, suggesting that the new GS2E allele has great potential in rice improvement. Taken together, we report new GS2 germplasm and describe a novel gene-editing strategy that can be widely employed to improve grain size and yield in rice. This trait-improvement strategy could be applied to other genes containing miRNA target sites, in particular the conserved miR396-GRF/GIF module that governs plant growth, development and environmental response.

Castle in the Sky: Dynamic Sky Replacement and Harmonization in Videos.

We propose a vision-based framework for dynamic sky replacement and harmonization in videos. Different from previous sky editing methods that either focus on static photos or require real-time pose signal from the camera's inertial measurement units, our method is purely vision-based, without any requirements on the capturing devices, and can be well applied to either online or offline processing scenarios. Our method runs in real-time and is free of manual interactions. We decompose the video sky replacement into several proxy tasks, including motion estimation, sky matting, and image blending. We derive the motion equation of an object at infinity on the image plane under the camera's motion, and propose "flow propagation", a novel method for robust motion estimation. We also propose a coarse-to-fine sky matting network to predict accurate sky matte and design image blending to improve the harmonization. Experiments are conducted on videos diversely captured in the wild and show high fidelity and good generalization capability of our framework in both visual quality and lighting/motion dynamics. We also introduce a new method for content-aware image augmentation and proved that this method is beneficial to visual perception in autonomous driving scenarios. Our code and animated results are available at https://github.com/jiupinjia/SkyAR.

Toward the Treatment of Inherited Diseases of the Retina Using CRISPR-Based Gene Editing.

Inherited retinal dystrophies [IRDs] are a common cause of severe vision loss resulting from pathogenic genetic variants. The eye is an attractive target organ for testing clinical translational approaches in inherited diseases. This has been demonstrated by the approval of the first gene supplementation therapy to treat an autosomal recessive IRD, RPE65-linked Leber congenital amaurosis (type 2), 4 years ago. However, not all diseases are amenable for treatment using gene supplementation therapy, highlighting the need for alternative strategies to overcome the limitations of this supplementation therapeutic modality. Gene editing has become of increasing interest with the discovery of the CRISPR-Cas9 platform. CRISPR-Cas9 offers several advantages over previous gene editing technologies as it facilitates targeted gene editing in an efficient, specific, and modifiable manner. Progress with CRISPR-Cas9 research now means that gene editing is a feasible strategy for the treatment of IRDs. This review will focus on the background of CRISPR-Cas9 and will stress the differences between gene editing using CRISPR-Cas9 and traditional gene supplementation therapy. Additionally, we will review research that has led to the first CRISPR-Cas9 trial for the treatment of CEP290-linked Leber congenital amaurosis (type 10), as well as outline future directions for CRISPR-Cas9 technology in the treatment of IRDs.

Advancements of CRISPR/Cas9 technology and its value in antiviral therapeutics

CISPR/Cas9 system is a natural immune mechanism adopted by bacteria and archaea on exposure to invading phages and plasmids. The field of genome editing has been revolutionized with the advent of CRISPR/Cas9 technology. The CRISPR/Cas9 based gene editing has offered a promising therapeutic platform for many animal and human diseases, particularly viral diseases because viruses evolve constantly and hence escape vaccine-induced immunity. The targeted genome editing by RNA-guided nucleases is rapid, easy, economical, and efficient compared to previous editing technologies. It not only helps in the direct destruction of viruses, but also helps us understand the host-virus interactions, gene functions, and develop recombinant vaccines. It has been widely experimented in the field of antiviral therapy, starting with HIV in 2013 to SARS CoV-2 recently, with a series of modifications in structure and composition of CRISPR/Cas9 and delivery mechanisms to achieve the ever-increasing promising results. Herein, this review focused on the origin of CRISPR/Cas9 system, mechanism of action, advantages over existing gene-editing tools, its progress in antiviral therapy, vaccine development, delivery approaches, and challenges faced in the application of CRISPR/Cas9.

Unlocking loxP to Track Genome Editing In Vivo.

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells

AbstractLentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing–based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Signal enhancement of glutamine and glutathione by single-step spectral editing

A single-step spectral editing approach using an always-on editing pulse was proposed to enhance the signals of strongly coupled spins. Specifically, a single-step spectral editing sequence with an always-on editing pulse applied at 2.12 ppm was used to enhance glutamine (Gln) and glutathione (GSH) signals at TE = 56 ms on a 7 T scanner. Density matrix simulations demonstrated that the current method (TE = 56 ms) led to large signal enhancement of at least 61% for Gln and 51% for GSH compared to a previous single-step method (TE = 106 ms). Monte Carlo simulations showed that the current method reduced noise-originated variations by 31% for Gln and 26% for GSH compared to a previous three-step spectral editing method from which the present method was derived.