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

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.

• Publication year

  2015

• Venue

  Genetics

• Publication date

  2015-07-17

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1534/genetics.115.179382PMID 26187122PMCID 4566275
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Rapid reverse genetic screening using CRISPR in zebrafish

  2015cited by this paper
• Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.

  2015cited by this paper
• Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

  2015cited by this paper
• Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

  2015cited by this paper
• Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

  2015cited by this paper
• CRISPR/Cas9 Genome Editing in Caenorhabditis elegans: Evaluation of Templates for Homology-Mediated Repair and Knock-Ins by Homology-Independent DNA Repair

  2015cited by this paper
• Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis

  2015cited by this paper
• Landscape of target:guide homology effects on Cas9-mediated cleavage

  2014cited by this paper
• Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

  2014influential reference
• Development and applications of CRISPR-Cas9 for genome engineering.

  2014cited by this paper
• Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

  2014cited by this paper
• Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

  2014influential reference
• The new frontier of genome engineering with CRISPR-Cas9

  2014cited by this paper
• Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

  2014cited by this paper
• Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus

  2014cited by this paper
• A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans

  2014cited by this paper
• Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation

  2014cited by this paper
• DNA targeting specificity of RNA-guided Cas9 nucleases

  2013cited by this paper
• Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas

  2013cited by this paper
• Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins

  2013influential reference
• Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination

  2013cited by this paper
• A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

  2012cited by this paper
• CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III

  2011cited by this paper
• Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene.

  1997influential reference
• Additional References I.

  1969cited by this paper

• Tagging of C. elegans apoptosis activator EGL-1 BH3-only reveals CED-9 BCL-2-dependent mitochondrial localization and dynamic control of EGL-1 synthesis and degradation in vivo.

  2026cites this paper
• Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders.

  2026cites this paper
• A developmental timer coordinates organism-wide microRNA transcription

  2026cites this paper
• A pilot study for whole proteome tagging in C. elegans

  2026influential citation
• Coordinated control of proteasome subunit gene expression promotes stress resistance, proteostasis, and longevity.

  2026cites this paper
• 4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.

  2025cites this paper
• Modulation by NPY/NPF-like receptor underlies experience-dependent, sexually dimorphic learning

  2025cites this paper
• Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo

  2025cites this paper
• Differential contributions of β-tubulin isotypes to acentrosomal oocyte meiosis in C. elegans

  2025cites this paper
• BAF-1–VRK-1 mediated release of meiotic chromosomes from the nuclear periphery is important for genome integrity

  2025cites this paper
• Notch controls APC/CFZR-1 to enable accumulation of chromatin regulators in germline stem cells from Caenorhabditis elegans

  2025cites this paper
• Protein sequence editing defines distinct and overlapping functions of SKN-1A/Nrf1 and SKN-1C/Nrf2

  2025cites this paper
• Oncogenic mutant KRAS inhibition through oxidation at cysteine 118

  2025cites this paper
• The β integrin modulates serotonin sensitivity via NPxY motifs to regulate egg laying and mechanosensation behaviors in C. elegans

  2025cites this paper
• Identification of an uncharacterized gene as a mitochondrial methionine tRNA synthetase in Caenorhabditis elegans

  2025cites this paper
• Loss of meiotic double strand breaks triggers recruitment of recombination-independent pro-crossover factors in C. elegans spermatogenesis

  2025cites this paper
• An ancient and essential miRNA family controls cellular interaction pathways in C. elegans

  2025cites this paper
• Distinct D-box Motifs in SPD-2 Mediate APC/CFZR−1-Dependent Degradation and Centrosomal Localization in Caenorhabditis elegans Embryos

  2025cites this paper
• Anticancer bioactive phytochemicals screening from medicinal-edible homologous materials in Caenorhabditis elegans.

  2025cites this paper
• MLT-11 is necessary for C. elegans embryogenesis and conserved sequences play distinct roles in cuticle structure

  2025cites this paper
• Tau clearance reverses touch neuron dysfunction in both young and aged C. elegans

  2025cites this paper
• Integrin is required for basement membrane crossing and branching of an invading intracellular tube.

  2025cites this paper
• The KASH protein UNC-83 differentially regulates kinesin-1 activity to control developmental stage-specific nuclear migration

  2025cites this paper
• Tau Clearance Reverses Neuronal Dysfunction in Both Young and Aged C. elegans

  2025cites this paper
• RG motifs promote piRNA-mediated gene silencing in C. elegans

  2025cites this paper
• Intergenerational transport of double-stranded RNA in C. elegans can limit heritable epigenetic changes

  2025cites this paper
• Condensate-forming eIF4ET ensures adequate levels of meiotic proteins to support oocyte storage

  2025cites this paper
• Food sensing controls C. elegans reproductive behavior by neuromodulatory disinhibition

  2025cites this paper
• A higher order PUF complex is central to regulation of C. elegans germline stem cells

  2025cites this paper
• Insertion of mNeonGreen into the variable domain of DRP-1 permits visualization of functional endogenous protein

  2025cites this paper
• Protein sequence editing defines distinct and overlapping functions of SKN-1A/Nrf1 and SKN-1C/Nrf2

  2025cites this paper
• Loss of Meiotic Double Strand Breaks Triggers Recruitment of Recombination-independent Pro-crossover Factors in C. elegans Spermatogenesis

  2025cites this paper
• C. elegans SSNA-1 is required for the structural integrity of centrioles and bipolar spindle assembly

  2025cites this paper
• Nutrient-Responsive Formation of Mitochondrial-Derived Structures in Caenorhabditis elegans

  2025cites this paper
• The mitochondrial trans-2-enoyl-coA reductase is necessary for mitochondrial homeostasis in C. elegans.

  2025cites this paper
• An SRR1 domain-containing protein is required for efficient Orsay virus replication in Caenorhabditis elegans

  2025cites this paper
• Three lncRNAs promote PUM protein condensation and germline differentiation

  2025cites this paper
• 不同秀丽隐杆线虫U6启动子对CRISPR/Cas9介导的基因编辑效率的影响

  2025cites this paper
• 3’ Nucleotide Asymmetry Directs miRNA Strand Selection

  2025cites this paper
• Effect of Caenorhabditis elegans U6 Promoters on CRISPR/Cas9-Mediated Gene Editing Efficiency

  2025cites this paper
• A chromatin-associated pool of Aurora A controls kinetochore-microtubule attachments to ensure chromosome biorientation

  2025cites this paper
• Multi-step implementation of meiotic crossover patterning

  2025cites this paper
• The MAST kinase KIN-4 carries out mitotic entry functions of Greatwall in C. elegans

  2025influential citation
• Redox homeostasis in ferroptosis and aging: a causal role for fard-1 and dhs-25 in Caenorhabditis elegans

  2025cites this paper
• Loss of an uncharacterized mitochondrial methionine tRNA-synthetase induces mitochondrial unfolded protein response in Caenorhabditis elegans

  2025cites this paper
• Adenosine deaminase and deoxyadenosine regulate intracellular immune response in C. elegans

  2025cites this paper
• A FMRF-amide peptide that regulates cell non-autonomous protein homeostasis in C. elegans

  2025cites this paper
• Lysine deficiency within a conserved lysine desert is critical for EEL-1/HUWE1 to support ubiquitin proteasome system function

  2025cites this paper
• Synthetic guide sequence to generate CRISPR-Cas9 entry strains in C. elegans

  2025cites this paper
• Viral RNA pUGylation Promotes Antiviral Immunity in C. elegans

  2025cites this paper
• Spatiotemporal single-cell architecture of gene expression in the Caenorhabditis elegans germ cells

  2025cites this paper
• Kinetochore components are perinuclear and form condensates in C. elegans germline

  2025cites this paper
• C. elegans septins regulate a subset of sensory neuronal cilia via cell-non autonomous mechanisms in supporting glia

  2025cites this paper
• Roles and regulation of the Kunitz domain protein MLT-11 during C. elegans cuticle synthesis and molting.

  2025cites this paper
• Damage-induced phosphorylation of BRC-1/BRD-1 in meiosis preserves germline integrity

  2025cites this paper
• Engineering the C. elegans genome with a nested, self-excising selection cassette

  2025cites this paper
• Exogenous expression of a histone H3.3 isoform causes extranuclear divisions in the C. elegans intestine

  2025cites this paper
• Design of lipid nanoparticle (LNP) containing genetic material CRISPR/Cas9 for familial hypercholesterolemia

  2025cites this paper
• Biased regulation of protein synthesis and hypoxic death by a conditional raptor mutation.

  2025cites this paper
• Cleavage of the Meckel-Gruber syndrome protein TMEM67 by ADAMTS9 uncouples Wnt signaling and ciliogenesis

  2025cites this paper
• Multiscale patterning of a model apical extracellular matrix revealed by systematic endogenous protein tagging

  2025cites this paper
• RG motifs promote piRNA-mediated gene silencing in C. elegans

  2025cites this paper
• The forkhead transcription factor FKH-7/FOXP acts in chemosensory neurons to regulate developmental decision-making

  2025cites this paper
• Cross-tissue coordination between SLC nucleoside transporters regulates reproduction in Caenorhabditis elegans

  2025cites this paper
• Efficient iterative CRISPR/Cas9 editing using sid-1 co-conversion and feeding RNAi in Caenorhabditis elegans

  2025cites this paper
• Cloning-Free Targeting of Endogenous Loci to Generate Fluorescent Reporters in Medaka

  2025cites this paper
• Regulation of MORC-1 is key to the CSR-1–mediated germline gene licensing mechanism in C. elegans

  2025cites this paper
• PLAA/UFD-3 regulates P-bodies through its intrinsic disordered domain

  2025cites this paper
• A lysosomal surveillance response to stress extends healthspan

  2025cites this paper
• Multiscale characterization of Caenorhabditiselegans mutants to probe functional mechanisms of human actin pathological variants

  2025cites this paper
• Synthetic guide sequence to generate CRISPR-Cas9 entry strains in C. elegans

  2025cites this paper
• Structural insights into SSNA1 self-assembly and its microtubule binding for centriole maintenance

  2025cites this paper
• Tri-snRNP activity modulates tauopathy phenotypes.

  2025cites this paper
• Fluorescent protein tagging of C. elegans core apoptosis pathway components reveals mitochondrial localization of CED-9 Bcl-2, CED-4 Apaf1 and CED-3 Caspase in non-apoptotic and apoptotic cells

  2025cites this paper
• The KASH protein UNC-83 differentially regulates kinesin-1 activity to control developmental stage-specific nuclear migration.

  2025cites this paper
• A single-copy knock-in system: one plasmid to target all chromosomes in C. elegans

  2025cites this paper
• eIF4E assembly into C. elegans germ granules is essential for its repressive function

  2025cites this paper
• A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling.

  2025cites this paper
• Analysis in vivo using a new method, ARGO (Analysis of Red Green Offset), reveals complexity and cell-type specificity in presynaptic turnover of synaptic vesicle protein Synaptogyrin/SNG-1

  2025cites this paper
• The PUF RNA-binding protein, FBF-2, maintains stem cells without binding to RNA

  2025cites this paper
• Active maintenance of meiosis-specific chromosome structures in C. elegans by the deubiquitinase DUO-1

  2025cites this paper
• Nuclear Argonaute HRDE-1 Maintains Chromatin-Independent Transcriptional Silencing through a Transgenerational Small RNA Feedback Loop

  2025cites this paper
• Viral RNA pUGylation promotes antiviral immunity in C. elegans

  2025cites this paper
• Autism risk variants in DLG4 ortholog increase penetrance of uncommon individual behavioral trait in Caenorhabditis elegans

  2025cites this paper
• Nicotinic acetylcholine receptors function with adhesion molecule SAX-7 to reverse cell orientation during migration.

  2025cites this paper
• High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

  2025cites this paper
• Germline status and micronutrient availability regulate a somatic mitochondrial quality control pathway via short-chain fatty acid metabolism

  2024cites this paper
• The kinase ZYG-1 phosphorylates the cartwheel protein SAS-5 to drive centriole assembly in C. elegans

  2024cites this paper
• Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis

  2024cites this paper
• The NHR-23-regulated putative protease inhibitor mlt-11 gene is necessary for C. elegans cuticle structure and function

  2024cites this paper
• The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line

  2024cites this paper
• Analyses of Genetic Regulation of the Nervous System in the Nematode Caenorhabditis elegans.

  2024cites this paper
• The outer kinetochore components KNL-1 and Ndc80 complex regulate axon and neuronal cell body positioning in the C. elegans nervous system

  2024cites this paper
• Domain 3a mutation of VPS33A suppresses larval arrest phenotype in the loss of VPS45 in Caenorhabditis elegans

  2024cites this paper
• Endogenous fluorescent reporters for heat shock proteins are not detectable after stress induction

  2024cites this paper
• PACS-1 variant protein is aberrantly localized in C. elegans model of PACS1/PACS2 syndromes

  2024cites this paper
• EDC-3 and EDC-4 Regulate Embryonic mRNA Clearance and Biomolecular Condensate Specialization

  2024cites this paper
• Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans

  2024cites this paper
• C. elegans: a prominent platform for modeling and drug screening in neurological disorders

  2024cites this paper
• Ribosome-Associated Vesicles promote activity-dependent local translation

  2024cites this paper