Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Chen, Xiangyang; Xu, Fei; Zhu, Chengming; Ji, Jiaojiao; Zhou, Xufei; Feng, Xuezhu; Guang, Shouhong

doi:10.1038/srep07581

Cited by 125 publications

(139 citation statements)

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“…Interestingly, these species have different modes of reproduction (C. briggsae, androdioecy vs. C. nigoni, gonochoristic) but can produce fertile hybrid progeny (Woodruff et al 2010), suggesting that H3K9me2 and H3K9me3 are interchangeable for X chromosome silencing. These findings are also in concordance with functional analyses of the chromatin remodeling NURF complex, where NURF is required for the sperm/oocyte decision in C. briggsae but not in C. nigoni (Chen et al 2014). Thus, germ-line chromatin marks have the capacity to evolve rapidly.…”

Section: H3k9me2 Vs H3k9me3supporting

confidence: 77%

“…Alternatively, Cbr-met-2(RNAi) may have had off-target effects, resulting in removal of both H3K9me2 and H3K9me3. To distinguish between these possibilities, we generated deletion mutations of Cbr-met-2 using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 with dual sgRNAs as described for C. elegans (Chen et al 2014) (Figure 5A). We isolated two independent deletions: Cbr-met-2(xoe1) and Cbr-met-2(xoe2).…”

Section: The X Chromosome Of Males Is Enriched For Different Repressimentioning

confidence: 99%

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Plasticity in the Meiotic Epigenetic Landscape of Sex Chromosomes inCaenorhabditisSpecies

Larson

Van

Nakayama³

et al. 2016

Genetics

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During meiosis in the heterogametic sex in some species, sex chromosomes undergo meiotic sex chromosome inactivation (MSCI), which results in acquisition of repressive chromatin and transcriptional silencing. In Caenorhabditis elegans, MSCI is mediated by MET-2 methyltransferase deposition of histone H3 lysine 9 dimethylation. Here we examined the meiotic chromatin landscape in germ lines of four Caenorhabditis species; C. remanei and C. brenneri represent ancestral gonochorism, while C. briggsae and C. elegans are two lineages that independently evolved hermaphroditism. While MSCI is conserved across all four species, repressive chromatin modifications are distinct and do not correlate with reproductive mode. In contrast to C. elegans and C. remanei germ cells where X chromosomes are enriched for histone H3 lysine 9 dimethylation, X chromosomes in C. briggsae and C. brenneri germ cells are enriched for histone H3 lysine 9 trimethylation. Inactivation of C. briggsae MET-2 resulted in germ-line X chromosome transcription and checkpoint activation. Further, both histone H3 lysine 9 di-and trimethylation were reduced in Cbr-met-2 mutant germ lines, suggesting that in contrast to C. elegans, H3 lysine 9 di-and trimethylation are interdependent. C. briggsae H3 lysine 9 trimethylation was redistributed in the presence of asynapsed chromosomes in a sex-specific manner in the related process of meiotic silencing of unsynapsed chromatin. However, these repressive marks did not influence X chromosome replication timing. Examination of additional Caenorhabditis species revealed diverse H3 lysine 9 methylation patterns on the X, suggesting that the sex chromosome epigenome evolves rapidly.

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Section: H3k9me2 Vs H3k9me3supporting

confidence: 77%

Section: The X Chromosome Of Males Is Enriched For Different Repressimentioning

confidence: 99%

Plasticity in the Meiotic Epigenetic Landscape of Sex Chromosomes inCaenorhabditisSpecies

Larson

Van

Nakayama³

et al. 2016

Genetics

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“…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).…”

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confidence: 99%

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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“…However, there are no reports concerning Cas9-mediated genome rearrangements in C. elegans. Our previous work showed that large chromosome fragments of up to 24 kb can be eliminated through the co-injection of two sgRNAs in C. elegans (Chen et al 2014). Here, we report the use of dual sgRNA-guided Cas9 nuclease to direct reciprocal chromosomal translocations in C. elegans.…”

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confidence: 99%

“…sgRNA #2 targeting exon 2 of the dpy-13 gene exhibited the highest efficiency and was used in the translocation experiments. For rde-12, the sgRNA targeting exon 2 of rde-12 was previously reported to have a high cleavage efficiency (Chen et al 2014) and was used in this work.We co-injected sgRNAs targeting rde-12 and dpy-13 with Cas9 and mCherry expression plasmids into eri-1(mg366) animals ( Figure S2). The mutation of eri-1 results in an enhanced RNAi (Eri) phenotype that facilitates the analysis of …”

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confidence: 99%

Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans

2015

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Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-offunction alleles of essential genes with detailed genetic background information.KEYWORDS CRISPR/Cas9; chromosomal translocation; essential gene knockout; balancer E SSENTIAL genes are required for the development and fertility of organisms. Loss-of-function mutations of essential genes usually result in growth arrest or sterility. The production and maintenance of homozygous mutants of the essential genes are demanding and time-consuming. A series of strains with particular chromosomal rearrangements, such as duplications, translocations, and inversions, have been generated and applied to screen and grow lethal or sterile mutants. Conventional methods to elicit chromosomal rearrangements involved treating the animals with ion irradiation or chemical mutagens (Jones et al. 2011). However, the majority of existing balancer strains lack detailed sequence information. Additionally, many unintended mutations are introduced during mutagenesis and are difficult to eliminate by backcrossing with wild-type strains. Therefore, it is critical to develop a more efficient method to produce balancer strains with detailed sequence information and nominal background mutations. Furthermore, the development of more effective approaches to generate and maintain loss-offunction mutations of particular essential genes is required.Recent research in targeted genome editing has made inspiring progress in genome engineering, among which is the clustered regularly interspaced short palindromic repeats (CRISPR) technology (Cong et al. 2013;Jiang et al. 2013;Mali et al. 2013;Ran et al. 2013;Wang et al. 2013;Hsu et al. 2014;Shalem et al. 2014;Sternberg et al. 2014;Wang et al. 2014). In the CRISPR/Cas9 system, small guide RNA (sgRNA) targets its complementary...

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Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Cited by 125 publications

References 58 publications

Plasticity in the Meiotic Epigenetic Landscape of Sex Chromosomes inCaenorhabditisSpecies

Plasticity in the Meiotic Epigenetic Landscape of Sex Chromosomes inCaenorhabditisSpecies

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

Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans

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