High Efficiency, Homology-Directed Genome Editing in <i>Caenorhabditis elegans</i> Using CRISPR-Cas9 Ribonucleoprotein Complexes

Paix, Alexandre; Folkmann, Andrew W.; Rasoloson, Dominique; Seydoux, Géraldine

doi:10.1534/genetics.115.179382

Cited by 652 publications

(590 citation statements)

References 23 publications

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“…We therefore examined changes in lamins during migration by imaging lamin tagged with GFP at the endogenous lmn-1 locus using CRISPR/Cas9-mediated genome editing Dickinson et al, 2013;Paix et al, 2015). C. elegans have a single lamin gene, lmn-1, that is broadly expressed and is essential for early embryonic divisions (Liu et al, 2000).…”

Section: P-cell Nuclei Undergo Dramatic Morphology Changes To Squeezementioning

confidence: 99%

Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells

et al. 2016

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Cellular migrations through constricted spaces are a crucial aspect of many developmental and disease processes including hematopoiesis, inflammation and metastasis. A limiting factor in these events is nuclear deformation. Here, we establish an in vivo model in which nuclei can be visualized while moving through constrictions and use it to elucidate mechanisms for nuclear migration. C. elegans hypodermal P-cell larval nuclei traverse a narrow space that is about 5% their width. This constriction is blocked by fibrous organelles, structures that pass through P cells to connect the muscles to cuticle. Fibrous organelles are removed just prior to nuclear migration, when nuclei and lamins undergo extreme morphological changes to squeeze through the space. Both actin and microtubule networks are organized to mediate nuclear migration. The LINC complex, consisting of the SUN protein UNC-84 and the KASH protein UNC-83, recruits dynein and kinesin-1 to the nuclear surface. Both motors function in P-cell nuclear migration, but dynein, functioning through UNC-83, plays a more central role as nuclei migrate towards minus ends of polarized microtubule networks. Thus, the nucleoskeleton and cytoskeleton are coordinated to move nuclei through constricted spaces.

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Section: P-cell Nuclei Undergo Dramatic Morphology Changes To Squeezementioning

confidence: 99%

Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells

et al. 2016

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“…Insertion events among the progeny can be identified only by PCR or by screening for the expected phenotype, which creates a large screening burden. There are two strategies to enrich for the rare animals with edited DNA: either by insertion of selectable markers at the target site or by co-CRISPR events at a second site (Arribere et al 2014;Kim et al 2014;Dickinson et al 2015;Norris et al 2015;Paix et al 2015;Ward 2015).…”

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confidence: 99%

“…The phenotype produced by the second-site edit identifies worms in which Cas9 was highly active, the progeny of which are enriched for edits at the target locus. However, among selected animals, the fraction of animals edited at the target locus is highly variable, so screening is still required (Arribere et al 2014;Paix et al 2015;Ward 2015). Hence, co-CRISPR strategies allow simplified repair template construction and allow target gene function in primary strains but reduce selection efficiency.…”

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confidence: 99%

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

2016

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In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cellspecific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3-to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.KEYWORDS CRISPR/Cas9; high-throughput; gene tagging; plasmid toolkit T HE clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized genome editing in nearly all model systems, including Caenorhabditis elegans (Frøkjaer-Jensen 2013;Doudna and Charpentier 2014;Xu 2015). The Cas9 protein cuts genomic DNA at sites that match an 20-nucleotide guide RNA sequence (Gasiunas et al. 2012;Jinek et al. 2012). Error-prone repair of the break can create point mutations, small indels, or large deletions at the cut site (Cho et al. 2013;Cong et al. 2013;Friedland et al. 2013;Jinek et al. 2013;van Schendel et al. 2015). However, the true power of CRISPR/Cas9 for genome editing lies in the insertion of exogenous DNA sequences, such as genetically encoded protein tags (Katic and Grosshans 2013;Lo et al. 2013;Mali et al. 2013;Tzur et al. 2013). To insert an exogenous sequence, one must simply supply a repair template with homology arms that flank the cut site. The cell uses homologybased repair to heal the break and copy the exogenous DNA into the cut site.Although in theory CRISPR/Cas9 makes it easy to insert exogenous sequences into a genome, practical limitations have prevented high-throughput implementation. Two critical limiting factors are (1) the time and expense required to build both the repair template a...

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“…Off-target cleavage has also been reduced by controlling the dosage of either the Cas9 protein or gRNA within the cell , or even by using Cas9 variants configured to enable conditional genome editing, such as a rapamycininducible split-Cas9 architecture (Zetsche et al 2015b) or a Cas9 variant that contains a strategically placed small-molecule-responsive intein domain . Nucleofection or transient transfection ) of a preformed Cas9 ribonucleoprotein complex has also been shown to reduce offtarget effects, enabling DNA-free gene editing in primary human T cells (Schumann et al 2015), embryonic stem cells (Liu et al 2015b), Caenorhabditis elegans gonads (Paix et al 2015), mouse (Menoret et al 2015;Wang et al 2015a) and zebrafish embryos , and even plant protoplasts (Woo et al 2015). The incorporation of specific chemical modifications known to protect RNA from nuclease degradation and stabilize secondary structure can further enhance Cas9 ribonucleoprotein activity (Hendel et al 2015;Rahdar et al 2015).…”

Section: Crispr-cas9mentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

297

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes

Cited by 652 publications

References 23 publications

Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells

Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

Genome-Editing Technologies: Principles and Applications

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