Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans

Huang, George Q.; Jesus, Bailey de; Koh, Alex; Blanco, Sara; Rettmann, Aubrie; DeMott, Ella; Sylvester, Melynda; Ren, Cassie; Meng, Carrie; Waterland, Skye; Rhodes, Anita; Alicea, Persephone; Flynn, Abbey; Dickinson, Daniel J.; Doonan, Ryan

doi:10.17912/micropub.biology.000460

Cited by 9 publications

(3 citation statements)

References 8 publications

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“…All strains were fed E-coli OP50 and maintained on nematode growth medium (NGM) plates at 20℃. Genetic modification including fluorescent protein knock-ins with/without degron AID was performed using CRISPR/Cas9-triggered homologous recombination following protocols published by our laboratory (Dickinson et al, 2013;Dickinson et al, 2015;Huang et al, 2021) Methods details…”

Section: Elegans Strain Construction and Maintenancementioning

confidence: 99%

Temporally distinct roles of Aurora A in polarization of theC. eleganszygote

Manzi,

de Jesus,

Shi

et al. 2023

Preprint

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During asymmetric cell division, coordination of cell polarity and the cell cycle is critical for proper inheritance of cell fate determinants and generation of cellular diversity. In Caenorhabditis elegans (C. elegans), polarity is established in the zygote and is governed by evolutionarily conserved Partitioning defective (PAR) proteins that localize to distinct cortical domains. At the time of polarity establishment, anterior and posterior PARs segregate to opposing cortical domains that specify asymmetric cell fates. Timely establishment of these PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C.elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including no posterior domain, reversed polarity, and excess posterior domains. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system, drug treatments, and high-resolution microscopy, we found that AIR-1 regulates polarity via distinct mechanisms at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent the formation of bipolar domains, while in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together these data clarify the origin of the multiple polarization phenotypes observed in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with the progression of the cell cycle.

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Section: Elegans Strain Construction and Maintenancementioning

confidence: 99%

Temporally distinct roles of Aurora A in polarization of theC. eleganszygote

Manzi,

de Jesus,

Shi

et al. 2023

Preprint

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“…The complex morphology and life cycles inherent to schistosomes are major hurdles in the efficient application of CRISPR/Cas9 for functional biology studies. Tactics developed for CRISPR/Cas9 studies on the model worm Caenorhabditis elegans [ 21 , 30 - 34 ], mammalian cells [ 35 - 39 ] and uni-cellular parasites [ 40 - 45 ] provide valuable information for exploring programmed genome editing in schistosomes [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase

Du,

McManus,

French

et al. 2023

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Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

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“…C. elegans is an ideal model system for genetics and molecular biology studies in vivo for numerous reasons: simple to manipulate and propagate, small and observable under a dissecting microscope, short generation cycle and lifespan, and ease of long-term frozen storage ( 8 ). There are numerous recent studies showing the value and utility of nematodes in engaging undergraduate students in inquiry and research through a course, including CUREs ( 9 – 15 ). In the GENE-CURE, students engage in comprehensive research, focusing on the structure-function analysis of disease-associated variants of uncertain significance (VUS) within human orthologs.…”

Section: Introductionmentioning

confidence: 99%

Engaging students in a genetics course-based undergraduate research experience utilizing Caenorhabditis elegans in hybrid learning to explore human disease gene variants

Forte,

Veasey,

Christie

et al. 2023

J Microbiol Biol Educ.

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Genetic analysis in model systems using bioinformatic approaches provides a rich context for a concrete and conceptual understanding of gene structure and function. With the intent to engage students in research and explore disease biology utilizing the nematode Caenorhabditis elegans model, we developed a semester-long course-based undergraduate research experience (CURE) in a hybrid (online/in-person) learning environment—the gene-editing and evolutionary nematode exploration CURE (GENE-CURE). Using a combination of bioinformatic and molecular genetic tools, students performed structure-function analysis of disease-associated variants of uncertain significance (VUS) in human orthologs. With the aid of a series of workshop-style research sessions, students worked in teams of two to six members to identify a conserved VUS locus across species and design and test a polymerase chain reaction-based assay for targeted editing of a gene in the nematode and downstream genotyping. Research session discussions, responsible conduct of research training, electronic laboratory notebook, project reports, quizzes, and group poster presentations at a research symposium were assessed for mastery of learning objectives and research progress. Self-reflections were collected from students to assess engagement, science identity, and science efficacy. Qualitative analysis of these reflections indicated several gains suggesting that all students found many aspects of the GENE-CURE rewarding (learning process of research, self-confidence in research and science identity, and personal interest) and challenging (iterative research and failure, time management, COVID-19 pandemic, and life issues).

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Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans

Cited by 9 publications

References 8 publications

Temporally distinct roles of Aurora A in polarization of theC. eleganszygote

Temporally distinct roles of Aurora A in polarization of theC. eleganszygote

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase

Engaging students in a genetics course-based undergraduate research experience utilizing Caenorhabditis elegans in hybrid learning to explore human disease gene variants

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