16 Papers found (0.01 seconds)

Ghanta KS et al. (2021) STAR Protoc "Microinjection for precision genome editing in Caenorhabditis elegans."

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STAR Protoc,

2021]

In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C.elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C.elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).

Ghanta KS et al. (2021) Elife "5' modifications improve potency and efficacy of DNA donors for precision genome editing."

[

Elife,

2021]

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Ghanta KS et al. (2020) Genetics "Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans."

Mello CC, Ghanta KS

[

Genetics,

2020]

CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

Ghanta, K.S et al. (2019) International Worm Meeting "5′ modified DNA donors for precision genome editing."

Ly, K, Krishnamurthy, P.M, Edraki, A, Mir, A, Watts, J.K, Sontheimer, E.J, Mello, C.C, Dokshin, G.A, Gneid, H, Ghanta, K.S

[

International Worm Meeting,

2019]

C.elegansis ideally suited for germline-editing, the syncytial germline allows each injection to deliver editing reagents to hundreds of germ nuclei. We recently described methodologies involving "asymmetric donors" (Dokshin et al, 2018), that yield one or more precision edits, such as GFP knock-ins per injected animal. Asymmetric donors are made by annealing a PCR product containing homology arms with an armless PCR product (containing, for example, gfp sequence only). We prefer the DNA marker Rol-6 as a metric and control for each of the many variables that influence editing efficiency. The presence of Rollers identifies a cohort of progeny inheriting long-dsDNA donors and edits. This cohort is not limited to the Rollers but also includes siblings of similar age to the Rollers. Scoring non-Rolling siblings is sometimes helpful when making a difficult edit. We do not prefer "co-editing" strategies such as "co-CRISPR" and "co-Conversion" when using CRISPR RNPs as these markers, likely due to a broader distribution of RNPs, do not necessarily identify animals inheriting longer precision DNA edits. After adjusting RNP and donor DNA levels such that the recovery of F1 Rollers is achieved at approximately 25% of the efficiency obtained when injecting rol-6 DNA alone, we find that typically 10% of Rollers assayed have GFP insertions while shorter edits, such as Degron or Flag insertions are found in greater than 50% of Rollers assayed. Recently, we have begun exploring how editing efficiencies depend on other donor DNA properties including chemical modifications added to the 5? ends. These studies began with attempts to improve donor DNA nuclear uptake by adding a nuclear localization signal (NLS). These NLS donors increased the frequency of GFP knock-ins dramatically, similar to the over-all efficiencies observed with asymmetric donors. Strikingly, however, high knock-in efficiencies were achieved at very low concentrations of just 10 ng/ l, for 1kb long donors, suggesting that the end modifications greatly enhance donor potency. Upon further investigation we found that the NLS was not required for high efficiency editing. Instead, our findings suggest that increased potency is driven by both a Tetra-Ethylene-Glycol (TEG) linker and a 2?-O-Methyl RNA (used to attach the NLS). Unfortunately, short donor molecules prepared using commercially sourced TEG primers have not (as yet) shown any increased editing efficiency. We are now exploring the specific chemistries and mechanisms through which end-modified donors influence the efficiency and accuracy of homology driven repair, and whether combining end-modifications and asymmetry, can further stimulate efficiencies.

Girgenrath, J.K. et al. (2019) International Worm Meeting "Investigating the interaction between germline specific DEAD-box helicase VBH-1 and CSR-1."

Ghanta, K.S., Mello, C.C., Girgenrath, J.K.

[

International Worm Meeting,

2019]

The Argonaute CSR-1 localizes to the perinuclear germline nuage, P-granules where it is required for RNA-induced epigenetic activation (RNAa), a process thought to protect endogenous germline transcripts from RNA-induced epigenetic silencing (RNAe) (Seth et al., 2013). To better understand CSR-1 function we identified interacting proteins through immunoprecipitation and Mass-Spectrometry analysis. This analysis revealed many peptides corresponding to the Vasa-Bell homolog, VBH-1, a DEAD-box protein previously shown to localize in P-granules (Salinas et al., 2007). The C. elegans germline expresses a wealth of DEAD-box proteins with widely varying functions. These include the GLH-1,2,3 and 4 proteins that, like VBH-1, co-localize in P-granules but instead of CSR-1, preferentially engage distinct Argonaute proteins. Although the exact function of VBH-1 has not been determined, our preliminary data shows that vbh-1 null mutants are viable but have significantly reduced brood sizes at 20? and 25? suggesting that it promotes temperature-dependent fertility. Interestingly, point mutants with lesions in the DEAD-box (DQAD mutants) exhibit gain-of-function or possibly antimorphic phenotypes that compromise fertility even more severely than do complete null mutants. Neither the null nor the DQAD mutants exhibit defects in CSR-1 localization. Also, vbh-1 null mutants did not have compromised RNAa activity, suggesting it is not required for this function of CSR-1. Currently, experiments are being conducted to characterize the protein-expression levels of CSR-1 targets in vbh-1 mutants. In the future, VBH-1 cross-linking IP (CLIP) experiments will be carried out to characterize the mRNA-binding profile of VBH-1. In addition, we are profiling CSR-1-associated small RNAs in wild-type and VBH-1 mutants to determine whether VBH-1 promotes the robustness of mRNA targeting at elevated temperatures. Using data generated through these experiments, we hope to obtain a greater understanding of CSR-1 and VBH-1 function in promoting fertility.

Huang, George et al. (2021) MicroPubl Biol

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

[

MicroPubl Biol,

2021]

The self-excising cassette (SEC) knock-in approach uses hygromycin selection and a visible roller phenotype to identify knock-ins, followed by a heat-shock induced excision of these visible markers to yield a seamless insertion of a fluorescent protein into the genome (Dickinson et al. 2015). Compared to protocols that utilize Cas9 protein and linear DNA repair templates (Paix et al. 2015; Dokshin et al. 2018; Ghanta and Mello 2020), the plasmid-based SEC approach employs a simpler screening strategy but requires more worms to be injected (Dickinson and Goldstein 2016).

DeMott, Ella et al. (2021) MicroPubl Biol

Doonan, Ryan, DeMott, Ella, Dickinson, Daniel J

[

MicroPubl Biol,

2021]

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.

Allyson Whittaker et al. (2001) International C. elegans Meeting "Control of the backing and turning steps of male mating behavior of C. elegans."

Allyson Whittaker, Gary Schindelman, Shahla Gharib, Paul Sternberg

[

International C. elegans Meeting,

2001]

Behavior in its simplest form consists of an organism sensing the environment and executing a motor output based on those signals. The next level is coordination of distinct behaviors, that is, processing a number of signals from the environment and triggering the appropriate response. C. elegans male mating behavior provides an excellent model for studying how behavior is coordinated and also for furthering our understanding of sensory perception. Mating behavior in C. elegans consists of a series of sub-behaviors with the initiation of one step correlating with the repression of the previous step. Understanding coordination of these behaviors will require furthering our knowledge of the molecular and neural pathways through which the different sub-steps are controlled. We are currently focusing on two of the earliest steps in mating behavior, backing and turning. Efforts are being made to address how these steps are triggered and executed and how one step is repressed when the next step is initiated. Previous research has provided information about the sensory, inter and motorneurons as well as the neurotransmitters required for the execution of these steps (1,2,3). We are currently doing a genetic screen to isolate new mutations that affect male mating behavior. The design of the screen will also allow us to identify mutations in the hermaphrodite which disrupt mating behavior enabling us to determine the hermaphrodite signals which trigger male responses. Further characterization of previously isolated mutations affecting backing and turning will also be described. 1. Liu, K.S. and Sternberg, P.W., Sensory regulation of male mating behavior in Caenorhabditis elegans . Neuron 14, 79-89 (1995). 2. Liu, K.S., Male mating behavior in Caenorhabditis elegans Ph.D. thesis, California Institute of Technology, Pasadena, California U.S.A. (1995). 3. Loer, C.M. and Kenyon, C.J. Serotonin-deficient mutants and male mating-behavior in the nematode Caenorhabditis elegans . J. Neurosci. 13, 5407-5417.

Katic, I. et al. (2019) International Worm Meeting "A reagent toolkit for tagging genes with compound tags."

Towbin, B., Katic, I., Gro beta hans, H.

[

International Worm Meeting,

2019]

Precise editing using CRISPR/Cas9-induced DNA breaks and subsequent homology-directed repair (HDR) have become a routine part of C. elegans research. Emerging consensus in the field is that single-stranded oligonucleotides work as highest-efficiency HDR templates for creating small inserts or sequence changes, as well as for generating defined deletions. Several recent methods have achieved increased efficiency of precise insertions of larger fragments (Farboud et al., 2019; Paix et al., 2016; Dokshin, Ghanta et al., 2018), using double-stranded or partially single stranded templates and ribonucleoprotein complex delivery of CRISPR-Cas9. To support the increased use of longer endogenous tagging by CRISPR/Cas9 gene editing in C. elegans, we present a set of plasmids that can serve as templates for generating linear double- or partially single-stranded HDR templates that are selection marker independent. The plasmids each contain a fluorescent protein coding region, as well as affinity tags and/or the auxin-dependent degron domain (Zhang et al., 2015). We have used this kit routinely for >40 tagging experiments with a 98% success rate. The kit can easily be expanded to encompass additional fluorescent proteins or other simple tags. Farboud, B., A. F. Severson, and B. Meyer. 2019 Strategies for Efficient Genome Editing Using CRISPR-Cas9. Genetics 211: 431-457. https://doi.org/10.1534/genetics.118.301775 Paix, A., H. Schmidt, and G. Seydoux. 2016 Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acid Res. doi: 10.1093/nar/gkw502 Dokshin, G. A., K. S. Ghanta, K. M. Piscopo, and C. C. Mello, 2018 Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. Genetics 210: 781-787. https://doi.org/10.1534/ge- netics.118.301532 Zhang, L., J. D. Ward, Z. Cheng, and A. F. Dernburg, 2015. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142: 4374-4384. doi: 10.1242/dev.129635

Hirotsu T et al. (1997) International C. elegans Meeting "ISOLATION AND CHARACTERIZATION OF MALE MATING-DEFECTIVE MUTANTS"

Hirotsu T, Yamamoto M, Iino Y

[

International C. elegans Meeting,

1997]

Male mating behavior is the most complex behavior known in C. elegans. We are interested in the mechanism whereby this male-specific and innate behavior is genetically programmed. Since 1983 (J. Hodgkin, Genetics 103, 43), many male mating-defective mutants have been isolated in several labs. A majority of these mutants have morphological defects in the copulatory organs in the tail (called mab mutants). We have started another round of mutant screening aiming at isolation of new mating-defective mutants, especially those that are morphologically normal. We mutagenized the plg-1; him-5 strain and 39 mutants that fail to deposit the copulatory plug, which is deposited over the hermaphrodite vulva after sperm transfer by the plg-1 males, at a normal rate were isolated. Male mating behavior is composed of "response to contact", "turning", "vulva location", "spicule insertion" and "sperm transfer" (K.S. Liu and P.W. Sternberg, Neuron 14, 79, 1995). Fourteen of the mutants had defects in response to contact, 23 in turning, and 5 in vulva location. Some of them had defects in multiple steps. Twenty of the mutants had morphological defects in the tail. These morphological defects included fusion and deletion of rays and shortening of rays. Six of the mutants occasionally showed projection of the spicules, suggesting defects in the spicule insertion/retraction step. Further analysis with DiO staining revealed defects in the sensory neurons in some of the morphologically normal mutants. Two of the mutants totally failed to adsorb DiO into the head amphid neurons, suggesting defects like "cilium-structure mutants". Some other mutants showed staining of supernumerary cells around amphid cell bodies. Further analysis of the mutants is under way.

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