Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges

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.

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In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.117.309881 ◽

2017 ◽

Vol 37 (9) ◽

pp. 1741-1747 ◽

Cited By ~ 95

Author(s):

Alexandra C. Chadwick ◽

Xiao Wang ◽

Kiran Musunuru

Keyword(s):

Genome Editing ◽

Proprotein Convertase ◽

Base Editing ◽

Therapeutic Alternative

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Modeling High-grade serous ovarian carcinoma using a combination of in vivo fallopian tube electroporation and CRISPR-Cas9-mediated genome editing

Cancer Research ◽

10.1158/0008-5472.can-20-1518 ◽

2021 ◽

pp. canres.1518.2020

Author(s):

Katie Teng ◽

Matthew J Ford ◽

Keerthana Harwalkar ◽

YuQi Li ◽

Alain Sarabia Pacis ◽

...

Keyword(s):

Ovarian Carcinoma ◽

Genome Editing ◽

Fallopian Tube ◽

High Grade ◽

Serous Ovarian Carcinoma

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Loss of Protein C in Zebrafish Results in Impaired Neutrophil Migration after Tissue Injury

Blood ◽

10.1182/blood-2020-142541 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-22

Author(s):

Chia-Jui Ku ◽

Steven Grzegorski ◽

Jordan A. Shavit

Keyword(s):

Genome Editing ◽

Protein C ◽

Tissue Injury ◽

Neutrophil Migration ◽

Neutrophil Recruitment ◽

Coagulation Cascade ◽

Consumptive Coagulopathy ◽

Neonatal Lethality ◽

Independent Functions

Hemostasis is a natural protective process that developed to retain a circulating blood system, conferred by a complicated yet sophisticated balance of factors. Disturbances of this network result in thrombosis or hemorrhage. Among many well-characterized coagulation factors, protein C (PC) exhibits multifunctional roles including anticoagulant, cytoprotective, and anti-inflammatory activities. The importance of PC has been demonstrated not only by the increased risk of venous thrombosis in individuals with heterozygous deficiency, but also the observed neonatal lethality in patients. Knockout mice exhibit similar neonatal lethality, which has made it difficult to further study complete deficiency. The zebrafish is a vertebrate organism that is characterized by a powerful genetic system, prolific breeding, rapid and transparent development, and a well described and highly conserved coagulation cascade. Here we utilize genome editing to generate a null allele of the PC gene (proc) in zebrafish and discover that its loss not only impairs hemostatic balance, but also affects neutrophil recruitment to sites of tissue injury. Through examination of publicly available zebrafish genome sequence, we determined that the proc locus is duplicated in tandem, resulting in two closely adjacent copies with nearly identical sequences. We used CRISPR/Cas9 with two single guide RNAs flanking the entire locus to produce a 17.3 kilobase deletion that knocks out both copies of proc to produce a complete null mutation, verified by sequencing and quantitative PCR. proc-/- mutants survived well into adulthood, with ~50% lethality by seven months of age. The embryonic survival and accessibility enabled us to perform intravital microscopy to evaluate the hemostatic effects of PC deficiency. We used laser-induced endothelial injury on the posterior cardinal vein (PCV) at 3 days post fertilization (dpf), which typically results in rapid formation of an occlusive fibrin-rich thrombus. proc-/- mutants had an average time to occlusion of 60 seconds versus 13 seconds in controls (p < 0.0001), consistent with a consumptive coagulopathy, as previously seen in antithrombin III (at3) mutants. A transgenic background with fluorescently labeled fibrinogen showed that more than 95% of proc-/- mutants had spontaneous thrombi in the PCV, which was not present in controls. To assess the role of PC in inflammation, we used two different injury strategies, non-vascular tail transection and chemical treatment (copper sulfate), on 3 dpf zebrafish larvae. Staining for neutrophil granules revealed homing to the site of injury within 60-75 minutes. In proc-/- mutants we found an average 50% reduction in the number of neutrophils recruited to the site of injury yet counts in the caudal hematopoietic tissue (the site of larval hematopoiesis) were unchanged. Since protein S (PS) is a cofactor for PC anticoagulant function, we hypothesized that the consumptive coagulopathy, but not the neutrophil recruitment, would be PS-dependent. We used genome editing to disrupt the PS gene (pros1) and found that loss of PS also results in a mild consumptive coagulopathy, but spontaneous thrombus formation was less common in the PCV (25%) and was often in the heart instead (80%). Neutrophil recruitment was unaffected in pros1 mutants, and evaluation of double proc/pros1 mutants revealed no synergy in any of the phenotypes. In conclusion, PC and PS deficiency in zebrafish show some similarity to our previously reported model of AT3 deficiency, but the effects are less potent, allowing robust survival that enables in vivo analyses. Our data suggest that the thrombotic phenotypes of PC and PS deficiency are not identical, and display tissue-specific phenotypes. We also found evidence for PS-independent functions of PC in neutrophil migration. We speculate this is due to the role that PC plays in inflammation and signaling but cannot exclude a role in neutrophil extracellular trap (NET) formation. This model of complete proc-/- deficiency in an accessible organism will facilitate further in vivo study of PS-dependent and independent functions of PC, as well as interplay between the two factors. Disclosures Shavit: Bayer: Consultancy; Taked: Consultancy.

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Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

International Journal of Molecular Sciences ◽

10.3390/ijms23020837 ◽

2022 ◽

Vol 23 (2) ◽

pp. 837

Author(s):

Sudip Biswas ◽

Nancy J. Wahl ◽

Michael J. Thomson ◽

John M. Cason ◽

Bill F. McCutchen ◽

...

Keyword(s):

Genome Editing ◽

New Technologies ◽

Fluorescent Protein ◽

Peanut Butter ◽

Processing System ◽

Protoplast Isolation ◽

Sequencing Analysis ◽

Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

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Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽

10.3390/biom11101526 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1526

Author(s):

Joanna E. Kowalczyk ◽

Shreya Saha ◽

Miia R. Mäkelä

Keyword(s):

Genome Editing ◽

Plant Biomass ◽

Genetic Manipulation ◽

White Rot ◽

White Rot Fungus ◽

Detailed Knowledge ◽

Wild Type ◽

Low Frequencies ◽

Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

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