Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype
Sonia El Mouridi,Claire Lecroisey,Philippe Tardy,Marine Mercier,Alice Leclercq-Blondel,Nora Zariohi,Thomas Boulin +6 more
TLDR
This strategy transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes.Abstract:
CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.read more
Citations
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Journal ArticleDOI
The Caenorhabditis elegans Transgenic Toolbox
TL;DR: A comprehensive overview of transgenic methods in C. elegans is provided, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.
Journal ArticleDOI
Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag.
TL;DR: A CRISPR-Tag system using one to four highly active sgRNAs to specifically label protein-coding genes with a high signal-to-noise ratio for visualization by wide-field fluorescence microscopy is developed, which marks genes of interest with small repeat sequences to recruit dCas9-GFP.
Journal ArticleDOI
High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce ( Lactuca sativa)
TL;DR: Targeting NCED4 in a co-editing strategy could be used to enrich for germline-edited events simply by germinating seeds at high temperature, and editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency.
Journal ArticleDOI
Single-Copy Knock-In Loci for Defined Gene Expression in Caenorhabditis elegans
Carlos G. Silva-García,Anne Lanjuin,Caroline Heintz,Sneha Dutta,Nicole M Clark,William B. Mair +5 more
TL;DR: The SKI LODGE system provides a fast, economical, and effective approach for generating single-copy ectopic transgenes in C. elegans.
Journal ArticleDOI
Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.
TL;DR: Nested CRISPR is developed, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans.
References
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Journal ArticleDOI
The new frontier of genome engineering with CRISPR-Cas9
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Journal ArticleDOI
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more
TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
Journal ArticleDOI
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.
Bernd Zetsche,Jonathan S. Gootenberg,Omar O. Abudayyeh,Ian Slaymaker,Kira S. Makarova,Patrick Essletzbichler,Sara E. Volz,Julia Joung,John van der Oost,Aviv Regev,Aviv Regev,Eugene V. Koonin,Feng Zhang +12 more
TL;DR: In this paper, the authors characterized Cpf1, a putative class 2 CRISPR effector, which is a single RNA-guided endonuclease lacking tracrRNA and utilizes a T-rich protospacer-adjacent motif.
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more
TL;DR: It is found that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches.
Journal ArticleDOI
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
John G. Doench,Nicolo Fusi,Meagan E Sullender,Mudra Hegde,Emma W Vaimberg,Katherine F Donovan,Ian Smith,Zuzana Tothova,Zuzana Tothova,Craig B. Wilen,Robert C. Orchard,Herbert W. Virgin,Jennifer Listgarten,David E. Root +13 more
TL;DR: Recently devised sgRNA design rules are used to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results, and a metric to predict off-target sites is developed.