PAIX, Alexandre et al. (2011) International Worm Meeting "RNA localization in the germline."

Seydoux, Geraldine, PAIX, Alexandre

[International Worm Meeting, 2011]

Many genes in the C. elegans germline are regulated by postrancriptional mechanisms acting through 3' UTR sequences. We are interested in how this regulation manifests itself at the level of RNA localization. To address this question, we are developing an in situ hybridization protocol to localize mRNAs in the germline with subcellular resolution. We will present our initial results comparing mRNAs that are ubiquitously expressed and mRNAs that are post-transcriptionally regulated in the mitotic zone.

Paix A et al. (2017) Methods "Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans."

Seydoux G, Folkmann A, Paix A

[Methods, 2017]

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4 days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.

Paix, A. et al. (2017) International Worm Meeting "Genome editing using Cas9 ribonucleoprotein complexes and linear repair templates."

Seydoux, G., Paix, A., Folkmann, A.

[International Worm Meeting, 2017]

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4 days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline. In this workshop, we will present our method and the rules governing successful genome editing in C. elegans

Paix A et al. (2015) Genetics "High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes."

Folkmann A, Paix A, Rasoloson D, Seydoux G

[Genetics, 2015]

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Paix A et al. (2016) Nucleic Acids Res "Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs."

Seydoux G, Schmidt H, Paix A

[Nucleic Acids Res, 2016]

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.

Paix A et al. (2014) Genetics "Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans."

Paix A, Wang Y, Krause M, Smith J, Calidas D, Lee CY, Schmidt H, Seydoux G, Smith H, Lu T

[Genetics, 2014]

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

Huang, George et al. (2021) MicroPubl Biol

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

[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).

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

Voronina E et al. (2012) Development "The P granule component PGL-1 promotes the localization and silencing activity of the PUF protein FBF-2 in germline stem cells."

Seydoux G, Voronina E, Paix A

[Development, 2012]

In the C. elegans germline, maintenance of undifferentiated stem cells depends on the PUF family RNA-binding proteins FBF-1 and FBF-2. FBF-1 and FBF-2 are 89% identical and are required redundantly to silence the expression of mRNAs that promote meiosis. Here we show that, despite their extensive sequence similarity, FBF-1 and FBF-2 have different effects on target mRNAs. FBF-1 promotes the degradation and/or transport of meiotic mRNAs out of the stem cell region, whereas FBF-2 prevents translation. FBF-2 activity depends on the P granule component PGL-1. PGL-1 is required to localize FBF-2 to perinuclear P granules and for efficient binding of FBF-2 to its mRNA targets. We conclude that multiple regulatory mechanisms converge on meiotic RNAs to ensure silencing in germline stem cells. Our findings also support the view that P granules facilitate mRNA silencing by providing an environment in which translational repressors can encounter their mRNA targets immediately upon exit from the nucleus.

Aoki, Scott Takeo et al. (2013) International Worm Meeting "Structural characterization of the P-granule protein scaffold."

Kimble, Judith, Aoki, Scott Takeo

[International Worm Meeting, 2013]

C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation. References: 1. Updike, D., Strome, S. (2010) J Androl 31: 53-60. 2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740. 3. Kawasaki, I., et al. (1998) Cell 94: 635-645. 4. Kawasaki, I., et al. (2004) Genetics 167: 645-661. 5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948. 6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..