Heritable Custom Genomic Modifications in <i>Caenorhabditis elegans</i> via a CRISPR–Cas9 System

Tzur, Yonatan B; Friedland, Ari E.; Nadarajan, Saravanapriah; Church, George M.; Calarco, John A.; Colaiácovo, Mónica P.

doi:10.1534/genetics.113.156075

Cited by 130 publications

(123 citation statements)

References 31 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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“…To induce mutations likely to be molecular nulls, we utilized CRISPR-associated protein 9 (Cas9) technology Tzur et al 2013). We created the single guide RNA (sgRNA) plasmid pCL45 by replacing the unc-119 targeting site in the plasmid PU6::unc-119_sgRNA (Addgene) with a target site near the start of exon 4 on the opposite strand (59-ggaagaaaaatacgccaaggAGG-39).…”

Section: Crispr/cas9 Induced Mutation Of Spe-47mentioning

confidence: 99%

“…Because we could not assess the severity of the spe-47(hc198) mutation on overall protein function, we created insertion/ deletion mutations (indels) in spe-47 using CRISPR/Cas9 technology Tzur et al 2013). CRISPR/Cas9 employs both the Cas9 nuclease from Streptococcus pyogenes and a sgRNA containing a targeting sequence directed to the desired site in the genome.…”

Section: Spe-47(hc198) Is a Semidominant Mutation Compared To Crispr/mentioning

confidence: 99%

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans

et al. 2015

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Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed "the brake model." Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a b-strand that mediates MSP-MSP dimerization. Both N-and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.

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“…Techniques to precisely manipulate genomes exist for many model organisms (Storici et al 2003;Gratz et al 2013;Hwang et al 2013;Jiang et al 2013;Tzur et al 2013), but not all features of these approaches are easily portable to closely related species. The genus Saccharomyces is highly experimentally tractable, and laboratory strains of all seven natural species can be genetically manipulated Liti et al 2013).…”

mentioning

confidence: 99%

High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

2014

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Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the Haploid Engineering and Replacement Protocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.G ENOME editing is a precise and powerful tool to investigate basic genetic processes or to reprogram an organism's metabolism. Techniques to precisely manipulate genomes exist for many model organisms (Storici et al. 2003;Gratz et al. 2013;Hwang et al. 2013;Jiang et al. 2013;Tzur et al. 2013), but not all features of these approaches are easily portable to closely related species. The genus Saccharomyces is highly experimentally tractable, and laboratory strains of all seven natural species can be genetically manipulated Liti et al. 2013). Saccharomyces cerevisiae is the most well-known member of the genus due to its role in brewing (Lodolo et al. 2008), biofuels (Steen et al. 2008;Matsushika et al. 2009), winemaking (Peris et al. 2012, and baking, as well as a model system for the biological sciences (Botstein and Fink 2011). Other members of the genus are also used by humans in the form of interspecies hybrids, such as the S. cerevisiae 3 Saccharomyces kudriavzevii hybrids used to ferment some wines and Belgian beers (Peris et al. 2012) and the S. cerevisiae 3 Saccharomyces eubayanus (Libkind et al. 2011) hybrids found in the brewing of lager-style beers around the world. The Saccharomyces genus is also an emerging "model genus" for molecular evolution, and several experimentally tractable species are now used routinely in evolutionary genetics research (Hittinger 2013). Efficient genome editing of these diverse Saccharomyces yeasts would therefore provide new avenues of investigation for basic and applied research.One major reason for the popularity of S. cerevisiae as a model system is the availability of powerful genetic manipulation tools. One of these tools is the URA3 selection/counterselection system (Boeke et al. 1984). URA3 is a gene required for the de novo synthesis of uracil. Thus, the URA3 gene can be used as a selectable marker in ura3 strains by selecting for the ability to grow on synthetic media without uracil. The deactivation or replacement of URA3 can also be selected for using synt...

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Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

Cited by 130 publications

References 31 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

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans

High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

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