scholarly journals Unlocking LoxP to Track Genome Editing In Vivo

2021 ◽  
Author(s):  
William Gendron ◽  
Jeffrey Rubin ◽  
Brandon Simone ◽  
Stephen Ekker ◽  
Michael Barry ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Genome Editing ◽  
Gene Editing ◽  
Fluorescent Proteins ◽  
Cellular Level ◽  
Ease Of Use ◽  
Editing Event ◽  
Detection Techniques ◽  
Organ Systems

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 and quantitatively monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses at the end of study. Here we report a new CRISPR “footprinting” approach to activate luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior Cre-detector system and is designed for Cas editors able to target LoxP including gRNAs including SaCas9 and ErCas12a [1, 2]. 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 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 recombinase 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.

scholarly journals Unlocking loxP to Track Genome Editing In Vivo

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1204
Author(s):  
William A. C. Gendron ◽  
Jeffrey D. Rubin ◽  
Michael J. Hansen ◽  
Rebecca A. Nace ◽  
Brandon W. Simone ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Genome Editing ◽  
Gene Editing ◽  
Fluorescent Proteins ◽  
Cellular Level ◽  
Ease Of Use ◽  
Editing Event ◽  
Detection Techniques ◽  
Organ Systems

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.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution

2009 ◽  
Vol 19 (8) ◽  
pp. e228-e233 ◽  
Author(s):  
Hee Young Kang ◽  
Philippe Bahadoran ◽  
Itaru Suzuki ◽  
Didier Zugaj ◽  
Abdallah Khemis ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Cellular Level ◽  
Reflectance Confocal Microscopy

Advances in therapeutic application of CRISPR-Cas9

2019 ◽  
Vol 19 (3) ◽  
pp. 164-174 ◽  
Author(s):  
Jinyu Sun ◽  
Jianchu Wang ◽  
Donghui Zheng ◽  
Xiaorong Hu
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Malignant Tumor ◽  
Gene Editing ◽  
Genome Engineering ◽  
Therapeutic Application ◽  
Adaptive Immune ◽  
Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

scholarly journals Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy

2010 ◽  
Vol 51 (2) ◽  
pp. 830 ◽  
Author(s):  
Beatrice Bourghardt Peebo ◽  
Per Fagerholm ◽  
Catharina Traneus-Röckert ◽  
Neil Lagali
Keyword(s):  
Confocal Microscopy ◽  
Cellular Level ◽  
In Vivo Confocal Microscopy ◽  
Lymph Vessels

In Vivo Laser Scanning Confocal Microscopy of the Ocular Surface in Glaucoma

2014 ◽  
Vol 20 (3) ◽  
pp. 879-894 ◽  
Author(s):  
Leonardo Mastropasqua ◽  
Luca Agnifili ◽  
Rodolfo Mastropasqua ◽  
Vincenzo Fasanella ◽  
Mario Nubile ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Ocular Surface ◽  
Laser Scanning ◽  
Current Knowledge ◽  
Laser Scanning Confocal Microscopy ◽  
Cellular Level ◽  
Surgical Approaches ◽  
Imaging Method ◽  
Scanning Confocal Microscopy

AbstractOver the past decade, knowledge about the ocular surface in glaucoma has significantly increased through the use of in vivo laser scanning confocal microscopy (LSCM). This in vivo imaging method can show modifications at the cellular level induced by anti-glaucoma drugs on ocular surface structures and adnexa in the eye. High-quality images of the conjunctiva, cornea, limbus, meibomian glands, and lymphoid structures during therapy can be obtained. In addition, LSCM opened new fields of research on the patho-physiology of aqueous humor (AH) hydrodynamics in untreated, and in medically or surgically treated glaucomatous patients. In these conditions, an enhancement of the trans-scleral AH outflow contributed to clarification of the mechanism of action of different anti-glaucoma medications and surgical approaches. Finally, the use of LSCM represented a huge advance in evaluation of bleb functionality after filtration surgery, defining the hallmarks of AH filtration through the bleb-wall and distinguishing functional from nonfunctional blebs. Thus, signs seen with LSCM may anticipate clinical failure, guiding the clinician in planning the appropriate timing of the various steps in bleb management. In this review we summarize the current knowledge about in vivo LSCM of the ocular surface in glaucoma.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

scholarly journals Fluorescent in vivo editing reporter (FIVER): A novel multispectral reporter of in vivo genome editing

2020 ◽  
Author(s):  
Peter A. Tennant ◽  
Robert G. Foster ◽  
Daniel O. Dodd ◽  
Ieng Fong Sou ◽  
Fraser McPhie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Disorders ◽  
Genetic Diseases ◽  
Primary Cells ◽  
Genetic Changes ◽  
Organ Systems ◽  
Therapeutic Development ◽  
The Right ◽  
Small Molecule Modulators

AbstractAdvances in genome editing technologies have created opportunities to treat rare genetic diseases, which are often overlooked in terms of therapeutic development. Nonetheless, substantial challenges remain: namely, achieving therapeutically beneficial levels and kinds of editing in the right cell type(s). Here we describe the development of FIVER (fluorescent in vivo editing reporter) — a modular toolkit for in vivo detection of genome editing with distinct fluorescent read-outs for non-homologous end-joining (NHEJ), homology-directed repair (HDR) and homology-independent targeted integration (HITI). We demonstrate that fluorescent outcomes reliably report genetic changes following editing with diverse genome editors in primary cells, organoids and in vivo. We show the potential of FIVER for high-throughput unbiased screens, from small molecule modulators of genome editing outcomes in primary cells through to genome-wide in vivo CRISPR cancer screens. Importantly, we demonstrate its in vivo application in postnatal organ systems of interest for genetic therapies — retina and liver. FIVER will broadly help expedite the development of therapeutic genome surgery for many genetic disorders.

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