Academic journal article Genetics

A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis Elegans

Academic journal article Genetics

A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis Elegans

Article excerpt

ABSTRACT Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counter-selection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

SEQUENCE-speci fi c immunity mechanisms such as RNA interference (Voinnet 2001; Zamore 2001; Grishok and Mello 2002; Hannon 2002) and CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (Horvath and Barrangou 2010; Bhaya et al. 2011; Terns and Terns 2011; Wiedenheft et al. 2012) provide sophisticated cellular de- fense against invasive nucleic acids. Understanding how these defense systems work has enabled researchers to re- direct them at cellular targets, providing powerful tools for manipulating both gene expression and the cellular genome itself. The CRISPR-Cas9 system is a bacterial antiviral mech- anism that captures fragments of viral DNA in specialized genomic regions for reexpression as small-guide RNAs (sgRNAs) (Bhaya et al. 2011; Terns and Terns 2011; Wiedenheft et al. 2012). In bacterial cells Cas9-sgRNA complexes provide acquired immunity against viral pathogens (Bhaya et al. 2011; Terns and Terns 2011; Wiedenheft et al. 2012). When coexpressed along with an artificial sgRNA designed to target a cellular gene, the Cas9 nuclease has been shown to ef fi- ciently direct the formation of double-strand breaks at the corresponding target locus (Jinek et al. 2012). Though bac- terial in origin, this mechanism works efficiently even within the context of eukaryotic chromatin (Gilbert et al. 2013). Genome editing using CRISPR-Cas9 has recently been dem- onstrated in numerous organisms, providing a powerful new tool with rapidly growing-if not infinite-potential for diverse biological applications (Bassett et al. 2013; Chang et al. 2013; Cho et al. 2013a; Cong et al. 2013; Dicarlo et al. 2013; Dickinson et al. 2013; Feng et al. 2013, 2014; Friedland et al. 2013; Gratz et al. 2013; Jiang et al. 2013; Mali et al. 2013b; Wang et al. 2013; Ma et al. 2014; Yu et al. 2014; Zhou et al. 2014).

The CRISPR-Cas9 system has also been successfully applied to Caenorhabditis elegans. Methods that have been used to express Cas9 include mRNA injection and transgene- driven expression from a constitutive or an inducible pro- moter (Chen et al. 2013; Chiu et al. 2013; Cho et al. 2013b; Dickinson et al. 2013; Friedland et al. 2013; Katic and Grosshans 2013; Lo et al. 2013; Tzur et al. 2013; Waaijers et al. 2013; Zhao et al. 2014). The U6 promoter has been used to drive sgRNA expression (Chiu et al. 2013; Dickinson et al. 2013; Friedland et al. 2013; Katic and Grosshans 2013; Waaijers et al. 2013). The system has been used widely to produce small insertions and deletions (indels) that shift the reading frame of the target gene, often resulting in premature termi- nation of translation and loss-of-function phenotypes (Chiu et al. 2013; Cho et al. 2013b; Friedland et al. 2013; Lo et al. 2013; Waaijers et al. 2013). In addition, single-strand oligo- nucleotides have been used as donor molecules to precisely alter a target gene through homologous recombination (HR) (Zhao et al. 2014), and a selection scheme has been devel- oped that allows the HR-mediated insertion of large sequence tags such as GFP (Chen et al. …

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