CRISPR Genome Editing Technology Research Articles

The application of CRISPR genome editing technologies in the pathogenesis studies, diagnosis, prevention and treatment of infectious diseases.

The CRISPR genome editing technology shows great application prospects in gene manipulation and infectious disease research, and is of great value for effective control and cure of infectious diseases. It has been utilized to generate specific disease models in cells, organoids and animals, which provide great convenience for research into the molecular mechanisms associated with infectious diseases. CRISPR screening technology enables high-throughput identification of risk factors. New molecular diagnostic tools based on CRISPR offer a more sensitive and faster method for detecting pathogens. The use of CRISPR tools to introduce resistance genes or to specifically destroy risk genes and virus genomes is intended to help prevent or treat infectious diseases. This review discusses the application of CRISPR genome editing technologies in the construction of disease models, screening of risk factors, pathogen diagnosis, and prevention and treatment of infectious diseases, thereby providing a reference for follow-up research in pathogenesis, diagnosis, prevention and treatment of infectious diseases.

CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.

54 Large-Scale Crispr Functional Characterization of Genomic Elements in Rainbow Trout (Oncorhynchus mykiss)

Abstract The wealth of genetic information that is now available for many agricultural animal species has opened vast potential to apply this new knowledge to improve agricultural production and sustainability. The field, however, is now faced with the daunting task of trying to understand what is arguably the most complex coded system known to mankind. In biomedical model systems, extensive efforts over decades have used single candidate gene approaches to uncover the function of many protein-coding genes and non-coding RNAs, but how this information translates to agricultural animals and their production traits is often unknown. Not only is repeating this vast effort for every agricultural species not practical, the current pace of genomic discovery is woefully inadequate for investigating animal genomes and therefore a radically new approach is needed. Using rainbow trout (RBT, Oncorhynchus mykiss) as a novel agricultural animal experimental system we have initiated a large-scale functional genomics screen that aims to characterize the function of over 1,000 genomic elements in a large animal model. The initial screen will focus on understanding how different genomic elements regulate growth and environmental resilience in these fish by combining multiplex CRISPR genome editing technology and phenotypic screening approaches to identify novel allele combinations from families of edited fish. The research is a paradigm shift in our approach to characterizing gene function, moving away from candidate-based approaches to a more genomics view of gene function. This work will shed light on the complex genetic mechanisms underlying two important production traits in fish while helping to advance our understanding of large animal functional genomics.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth

Gliomas are the most prevalent primary tumor of the CNS. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 (HNK-1) capped O-Man core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of HNK-1 capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors

The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5%± 17.5% and 8.4%± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.

Genome-Wide Identification of the Aconitase Gene Family in Tomato (Solanum lycopersicum) and CRISPR-Based Functional Characterization of SlACO2 on Male-Sterility.

Tomato (Solanum lycopersicum) is one of the most cultivated vegetables in the world due to its consumption in a large variety of raw, cooked, or processed foods. Tomato breeding and productivity highly depend on the use of hybrid seeds and their higher yield, environmental adaption, and disease tolerance. However, the emasculation procedure during hybridization raises tomato seed production costs and labor expenses. Using male sterility is an effective way to reduce the cost of hybrid seeds and ensure cultivar purity. Recent developments in CRISPR genome editing technology enabled tomato breeders to investigate the male sterility genes and to develop male-sterile tomato lines. In the current study, the tomato Acotinase (SlACO) gene family was investigated via in silico tools and functionally characterized with CRISPR/Cas9-mediated gene disruption. Genome-wide blast and HMM search represented two SlACO genes located on different tomato chromosomes. Both genes were estimated to have a segmental duplication in the tomato genome due to their identical motif and domain structure. One of these genes, SlACO2, showed a high expression profile in all generative cells of tomato. Therefore, the SlACO2 gene was targeted with two different gRNA/Cas9 constructs to identify their functional role in tomatoes. The gene was mutated in a total of six genome-edited tomato lines, two of which were homozygous. Surprisingly, pollen viability was found to be extremely low in mutant plants compared to their wild-type (WT) counterparts. Likewise, the number of seeds per fruit also sharply decreased more than fivefold in mutant lines (10-12 seeds) compared to that in WT (67 seeds). The pollen shape, anther structures, and flower colors/shapes were not significantly varied between the mutant and WT tomatoes. The mutated lines were also subjected to salt and mannitol-mediated drought stress to test the effect of SlACO2 on abiotic stress tolerance. The results of the study indicated that mutant tomatoes have higher tolerance with significantly lower MDA content under stress conditions. This is the first CRISPR-mediated characterization of ACO genes on pollen viability, seed formation, and abiotic stress tolerance in tomatoes.

From Pharma to Farm Can CRISPR Feed the World?

From Pharma to Farm Can CRISPR Feed the World?

CRISPR-Cas knockout of miR21 reduces glioma growth

Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.

Antibody-mediated delivery of CRISPR-Cas9 ribonucleoproteins in human cells.

The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.

CRISPR gene editing of major domestication traits accelerating breeding for Solanaceae crops improvement.

Domestication traits particularly fruit size and plant architecture and flowering are critical in transforming a progenitor's wild stature into a super improved plant. The latest advancements in the CRISPR system, as well as its rapid adoption, are speeding up plant breeding. Solanaceae has a varied range of important crops, with a few model crops, such as tomato and, more recently, groundcherry, serving as a foundation for developing molecular techniques, genome editing tools, and establishing standards for other crops. Domestication traits in agricultural plants are quantified and widely adopted under modern plant breeding to improve small-fruited and bushy crop species like goji berry. The molecular mechanisms of the FW2.2, FW3.2, FW11.3, FAS/CLV3, LC/WUS, SP, SP5G, and CRISPR genome editing technology have been described in detail here. Furthermore, special focus has been placed on CRISPR gene editing achievements for revolutionizing Solanaceae breeding and changing the overall crop landscape. This review seeks to provide a thorough overview of the CRISPR technique's ongoing advancements, particularly in Solanaceae, in terms of domesticated features, future prospects, and regulatory risks. We believe that this vigorous discussion will lead to a broader understanding of CRISPR gene editing as a tool for achieving key breeding goals in other Solanaceae minor crops with significant industrial value.

CRISPR Tackles Emerging Viral Pathogens.

Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.

Increasing eDNA capabilities with CRISPR technology for real-time monitoring of ecosystem biodiversity.

The accelerating pace of scientific discovery is being driven by the development of transformative technologies that are expanding traditional scientific boundaries. In ecology, the concept that ecosystem structure can be monitored through the stew of genetic material released into the environment by local organisms is now a highly active area of research. At the same time that the use of environmental DNA (eDNA) has gained popularity in ecology, CRISPR genome editing technology has been revolutionizing organismal biology and the biomedical sciences. In a From the Cover article in this issue of Molecular Ecology Resources, Williams et al. (2019) report about the fusion of CRISPR technology with molecular ecology to improve the in situ processing capabilities of eDNA applications. Piloting this new CRISPR technology on aquatic systems, the authors describe tools to accurately identify the presence of Atlantic salmon (Salmo salar) from eDNA, using conditions that are highly amenable to implementation in the field. This overcomes a major barrier restricting the use of eDNA, opening a door to expanded use of the technology in ecological research.

Abstract 2153: Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology

Abstract Melanoma remains the most lethal form of skin cancer exhibiting high mortality rates due to a high likelihood of developing metastases and acquiring drug resistance. Approximately 40-50% of melanomas contain oncogenic BRAF mutations of which 75-90% harbor the BRAFV600E mutation. This mutation constitutively activates the mitogen-activating protein kinase (MAPK) signaling pathway leading to uncontrolled cell growth and oncogenesis. Recent combination therapies of BRAF and MEK specific inhibitors have shown improved progression-free patient response in phase II clinical trials. However, in the majority of patients, acquired resistance to MAPK pathway inhibitor therapies develops after approximately 12 months of treatment. Preclinical studies have suggested that MEK1 mutations confer resistance to BRAF and MEK inhibitors. In this study, we used the CRISPR genome editing technology to generate a drug resistant MEK1Q56P knock-in mutation within the A375 melanoma cell line which naturally harbors the BRAFV600E mutation. We validated this new isogenic cell model using both molecular and biofunctional approaches. Method and results: Single guide RNAs (sgRNAs) were designed and built into Cas9 plasmids to bind and cut desired regions in the MEK1 target region. The melanoma cell line A375 was co-transfected with single guide-containing Cas9 plasmids and donor plasmids. Single cells were cloned and expanded for subsequent screening of the desired gene mutation events. The introduction of the MEK1Q56P mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic level. Validation of the mutation in mRNA transcripts was carried out by RT-PCR followed by sequencing. The cell line maintains permanent and genetically stable resistance characteristics during cell culture expansion without the use of selective pressure. Drug responses to BRAF and MEK1 specific inhibitors and non-specific chemotherapy drugs were compared between the A375 MEK1Q56P isogenic cell line and the parental cell line in 2D and 3D culture environments. Results demonstrated that the isogenic MEK1Q56P cell line showed significant and specific resistance to BRAF inhibitors in comparison to the parental A375 line. Using gene editing we have targeted an endogenous loci within a melanoma cell line creating a novel in-vitro model to aid in basic and translational melanoma research as well as drug screening efforts. This new approach to cell line development provides direct in vitro bio-functional evidence of a drug resistant gene driving tumor cells survival under targeted anti-cancer treatments. Furthermore, this A375 MEK1Q56P isogenic cell line represents a new type of drug resistance model that contains a defined genetic resistance mechanism. This model provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma. Citation Format: Lysa-Anne Volpe, Metewo Selase Enuameh, Luping Chen, Elizabeth Turner, John Foulke, Fang Tian. Development of a novel MEK mutation driven drug resistant melanoma model by CRISPR/Cas9 technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2153.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Osr1 Interacts Synergistically with Wt1 to Regulate Kidney Organogenesis.

Renal hypoplasia is a common cause of pediatric renal failure and several adult-onset diseases. Recent studies have associated a variant of the OSR1 gene with reduction of newborn kidney size and function in heterozygotes and neonatal lethality with kidney defects in homozygotes. How OSR1 regulates kidney development and nephron endowment is not well understood, however. In this study, by using the recently developed CRISPR genome editing technology, we genetically labeled the endogenous Osr1 protein and show that Osr1 interacts with Wt1 in the developing kidney. Whereas mice heterozygous for either an Osr1 or Wt1 null allele have normal kidneys at birth, most mice heterozygous for both Osr1 and Wt1 exhibit defects in metanephric kidney development, including unilateral or bilateral kidney agenesis or hypoplasia. The developmental defects in the Osr1+/-Wt1+/- mouse embryos were detected as early as E10.5, during specification of the metanephric mesenchyme, with the Osr1+/-Wt1+/- mouse embryos exhibiting significantly reduced Pax2-positive and Six2-positive nephron progenitor cells. Moreover, expression of Gdnf, the major nephrogenic signal for inducing ureteric bud outgrowth, was significantly reduced in the metanephric mesenchyme in Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- littermates. By E11.5, as the ureteric buds invade the metanephric mesenchyme and initiate branching morphogenesis, kidney morphogenesis was significantly impaired in the Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- embryos. These results indicate that Osr1 and Wt1 act synergistically to regulate nephron endowment by controlling metanephric mesenchyme specification during early nephrogenesis.

Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C.albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C.albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C.albicans genetic manipulation.