CRISPR/Cas9-Targeted Mutagenesis in <i>Caenorhabditis elegans</i>

Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B. L. G.; Tijsterman, Marcel; Heuvel, Sander van den; Boxem, Mike

doi:10.1534/genetics.113.156299

Cited by 159 publications

(147 citation statements)

References 19 publications

(27 reference statements)

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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) 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.The median frequency of NHEJ-mediated mutagenesis for successfully edited C. elegans targets was 4.3% (range of 0.2-100%) in published studies using DNA sequence to screen targets in transgenic F 1 animals expressing Cas9 (Table S1) Waaijers et al 2013;Kim et al 2014). In contrast, the median frequency of editing for targets in our study was 51% (range of 10-72%) (Table S1), a 10-fold increase (P # 0.02.…”

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confidence: 58%

“…All guides designed for all targets supported robust genome editing, both imprecise NHEJ events and precise, templated HDR events. The median frequency of editing at all targets was 51% without any coselectable markers, a 10-fold increase above previous studies that also reported numerous guide RNAs in the nonfunctional class Waaijers et al 2013;Kim et al 2014). Combining our already effective guide RNA design with the co-conversion/ co-CRISPR strategy of others (Arribere et al 2014;Kim et al 2014;Ward 2014) enhanced the ease of mutant recovery and boosted our median for both precise and imprecise genome editing to 86%.…”

mentioning

confidence: 48%

“…The mutants did not need to have a visible phenotype. In all cases, shifting the 39 GG out of the protospacer universally reduced the frequency of mutagenesis, but to a variable and unpredictable level (median frequency 1%, range of 0-21%) reflective of the more common range of frequencies found in most C. elegans studies using non-39 GG guides and related screening approaches (Table S1) Waaijers et al 2013;Kim et al 2014).To understand the variability in success of the 39 GGshift guides, we compared our guide sequences and editing frequencies to those of other organisms. Analysis of guide RNAs found to be effective in zebrafish (Gagnon et al 2014), Drosophila (Ren et al 2014, and mammalian cell lines (Doench et al 2014;Wang et al 2014;Wu et al 2014) suggests that the partial success of some 39 GG-shift guides in C. elegans might be due either to a G at position 20 of the target-specific sequences of the guide or to any combination of two purines at positions 19 and 20.…”

mentioning

confidence: 96%

“…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%

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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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contrasting

confidence: 58%

mentioning

confidence: 48%

mentioning

confidence: 96%

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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“…Compared with the zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which have been used for genome editing [1], the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated (Cas) system has emerged as a new powerful tool for genome modifications. It has recently been adopted for genome editing in human cell lines [2][3][4], mouse [5], zebrafish [6], C. elegans [7][8][9][10][11][12], and plants [13].…”

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confidence: 99%

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

Zhao

Zhang

et al. 2014

Cell Res

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Konditionale Kontrolle der CRISPR/Cas9‐Funktion

Zhou

Deiters

2016

Angewandte Chemie

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Das CRISPR/Cas9‐Endonukleasesystem, das aus einer Guide‐RNA zur Erkennung einer Zielsequenz der DNA und dem Cas9‐Nukleaseprotein zur Bindung und Prozessierung der Ziel‐DNA besteht, wurde intensiv erforscht und findet bereits breite Anwendung im Gen‐Editing, der Synthesebiologie und der transkriptionellen Modulation in Zellen und Tieren. Mit dem Ziel einer präziseren Genmodifizierung und der Weiterentwicklung des CRISPR/Cas9‐Systems zu einem räumlich‐zeitlich kontrollierbaren Genregulationssystem wurden kürzlich mehrere Ansätze zur konditionalen Aktivierung der Cas9‐Funktion durch niedermolekulare Verbindungen (“kleine Moleküle”) oder Licht entwickelt. Diese Methoden führten zu einer verbesserten Spezifität des CRISPR/Cas9‐Systems im Gen‐Editing und ermöglichten dessen Aktivierung mit zeitlicher und räumlicher Präzision.

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CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans

Cited by 159 publications

References 19 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

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

Konditionale Kontrolle der CRISPR/Cas9‐Funktion

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