Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in<i>Caenorhabditis elegans</i>

Paix, Alexandre; Wang, Yuemeng; Smith, Harold E.; Lee, Chih Yung S.; Calidas, Deepika; Lu, Tu; Smith, Jarrett; Schmidt, Helen; Krause, Michael; Seydoux, Géraldine

doi:10.1534/genetics.114.170423

Cited by 308 publications

(372 citation statements)

References 26 publications

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“…Our efforts at Cas9-directed editing succeeded with all 39 GG guides. In contrast, success was infrequent and unpredictable with guides lacking a 39 GG in our study and in published studies (Table S1) (Chiu et al 2013;Cho et al 2013;Dickinson et al 2013;Friedland et al 2013;Katic and Grosshans 2013;Lo et al 2013;Tzur et al 2013;Liu et al 2014;Paix et al 2014;Shen et al 2014;Waaijers and Boxem 2014;Zhao et al 2014). We also found that a 39 GG dinucleotide reliably increased the frequency of genome editing at all targets, even related targets for which the 39 GG-shift guides supported a low to modest frequency of editing.…”

supporting

confidence: 56%

“…The initial report describing the successful use of Cas9 in C. elegans demonstrated a range of editing frequencies from 0.5 to 80%, with only two targets exceeding 4% . Subsequent publications also reported variably low mutagenesis rates that required molecular screening of hundreds of animals or required the desired mutation to cause an easily detectable mutant phenotype or to be introduced in tandem with a coselection marker (Chiu et al 2013;Cho et al 2013;Dickinson et al 2013;Katic and Grosshans 2013;Lo et al 2013;Tzur et al 2013;Waaijers et al 2013;Chen et al 2014;Liu et al 2014;Paix et al 2014;Shen et al 2014;Zhao et al 2014). More recent studies greatly improved the odds of detecting a targeted mutation through the simultaneous co-conversion of a mutation in an unrelated target that causes a visible phenotype (Arribere et al 2014;Kim et al 2014;Ward 2014).…”

mentioning

confidence: 99%

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Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Farboud¹,

2015

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Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 39 end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 39 GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome. KEYWORDS CRISPR; Cas9; genome editing; co-conversion; C. elegans T HE use of site-specific nucleases with programmable target specificity has transformed the art of genome editing and thereby revolutionized the dissection and manipulation of genome function (reviewed in Mali et al. 2013;Carroll 2014;Doudna and Charpentier 2014;Hsu et al. 2014). Most widely used is the CRISPR-associated nuclease Cas9, whose RNA-programmed DNA cleaving activities create DNA double-strand breaks (DSBs). These DSBs can be repaired imprecisely by nonhomologous end joining (NHEJ) to generate random insertions and deletions or repaired precisely by homology-directed repair (HDR) templated from exogenous DNA to generate custom-designed insertions, deletions, or substitutions (Gasiunas et al. 2012;Jinek et al. 2012;Cong et al. 2013;Jinek et al. 2013;Mali et al. 2013). Modified variants of Cas9 that lack DNA cleaving activity have also been utilized to regulate transcription of designated gene targets and to cytologically mark and track genomic loci in living cells (Bikard et al. 2013;Chen et al. 2013;Larson et al. 2013;Maeder et al. 2013;Perez-Pinera et al. 2013;Qi et al. 2013;Gilbert et al. 2014;Tanenbaum et al. 2014).The Cas9 protein is targeted to a specific genomic locus by a guide RNA that encodes a 20-nt region of homology to the DNA target ( Figure 1A) (Mojica et al. 2009;Garneau et al. 2010;Jinek et al. 2012). The most commonly used guide RNAs are chimeric fusions between the CRISPR RNA (crRNA), which encodes the 20-nt target-specific sequence, and the tracer RNA (trRNA), which enables the formation of active Cas9-guide RNA complexes ( Figure 1A) (Jinek et al. 2012). Few constraints are known for Cas9 ...

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supporting

confidence: 56%

mentioning

confidence: 99%

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Farboud¹,

2015

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“…Short homology arms of just 30-60 bp were pioneered for co-CRISPR applications by the Seydoux group (Paix et al 2014). We have similarly observed that homology arms of just 57 bp generated insertions at high frequency at most loci tested.…”

Section: Discussionmentioning

confidence: 70%

“…In previous studies, homology arms as short as 500 bp for plasmid repair templates and 30 bp for linear repair templates efficiently direct insertion into the C. elegans genome (Paix et al 2014;Dickinson et al 2015). To determine the effect of homology arm length on insertion frequency for SapTrap vectors, we targeted the 59 end of the snb-1 gene with a gfp cassette flanked by homology arms 0, 44, 100, or 400 bp in length ( Figure 3A and Table S3).…”

Section: Short Homology Armsmentioning

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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“…Recently, a CRISPR allele of nos-2 has also been created. 21 Both nos-2 mutations confer only a minor sterility on their own, in contrast to reports of nos-2 (RNAi). 8 Since nos-2(RNAi) should also knock down expression of the T02G5.11, it remains possible that this predicted pseudogene retains some ancestral function.…”

Section: Nanos Family Members In C Elegansmentioning

confidence: 82%

A twist of fate: How a meiotic protein is providing new perspectives on germ cell development

Rana

Yanowitz

2016

Worm

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The molecular pathways that govern how germ line fate is acquired is an area of intense investigation that has major implications for the development of assisted reproductive technologies, infertility interventions, and treatment of germ cell cancers. Transcriptional repression has emerged as a primary mechanism to ensure suppression of somatic growth programs in primordial germ cells. In this commentary, we address how xnd-1 illuminates our understanding of transcriptional repression and how it is coordinated with the germ cell differentiation program. We recently identified xnd-1 as a novel, early determinant of germ cell fates in Caenorhabditis elegans. Our study revealed that XND-1 is maternally deposited into early embryos where it is selectively enriched in the germ lineage and then exclusively found on chromatin in the germ lineage throughout development and into adulthood when it dissociates from chromosomes in late pachytene. This localization is consistent with a range of interesting germ cell defects that suggest xnd-1 is a pivotal determinant of germ cell characteristics. Loss of xnd-1 results in a unique "one PGC (primordial germ cell)" phenotype due to G2 cell cycle arrest of the germline precursor blastomere, P 4 , which predisposes the animal and its progeny for reduced fecundity. The sterility in xnd-1 mutants is correlated with an increase in the transcriptional activationassociated histone modification, dimethylation of histone H3 lysine 4 (H3K4me2), and aberrant expression of somatic transgenes but overlapping roles with nos-2 and nos-1 suggest that transcriptional repression is achieved by multiple redundant mechanisms.

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Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites inCaenorhabditis elegans

Cited by 308 publications

References 26 publications

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

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

A twist of fate: How a meiotic protein is providing new perspectives on germ cell development

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