Academic journal article Genetics

Rapid and Precise Engineering of the Caenorhabditis Elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

Academic journal article Genetics

Rapid and Precise Engineering of the Caenorhabditis Elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

Article excerpt

SEQUENCE-SPECIFIC nucleases are a critical tool for manipulation of DNA sequences. The bacterial type II clustered regularly interspaced short palindromic repeats (CRISPR) system, which normally protects against viral DNA and provides a memory of exposure (Jinek et al. 2012), has recently revolutionized genome editing in multiple organisms (Cong et al. 2013; DiCarlo et al. 2013; Gratz et al. 2013; Hwang et al. 2013; Li et al. 2013; Ran et al. 2013; Gratz et al. 2014; Nakanishi et al. 2014). For genome editing, the system has been simplified to two components: the Cas9 nuclease, which generates DNA double-strand breaks (DSBs), and a chimeric small guide RNA (sgRNA) that fills the function of two small RNAs in the native bacterial system (Cong et al. 2013). Specific genomic sequences are targeted by the 59-most 15-20 bp of the sgRNA through the formation of an RNA:DNA hybrid (Jinek et al. 2012; Mali et al. 2013). An NGG motif (protospacer adjacent motif, PAM) must immediately follow the target sequence in the genome (Jinek et al. 2012; Ran et al. 2013). This PAM directs Cas9 to cleave the DNA 3 bp 59 to the PAM (Jinek et al. 2012). Depending on the desired experimental outcome, one can select for error-prone repair by pathways such as nonhomologous end joining (NHEJ) to generate insertion-deletion (indel) mutations, or homologous recombination to knock in specific sequences.

Initial genome editing methods in Caenorhabditis elegans harnessed excision of a Tc or Mos transposon to generate a DSB, and a plasmid repair template to knock in (Plasterk and Groenen 1992; Robert and Bessereau 2007; FrøkjaerJensen et al. 2008; Frøkjær-Jensen et al. 2012), or delete (Frøkjær-Jensen et al. 2010) desired sequences through homologous recombination. These methods are robust, but the relative rarity of the editing event requires use of a selectable marker, such as unc-119 rescue or antibiotic resistance (Frøkjaer-Jensen et al. 2008; Giordano-Santini et al. 2010), and a transposon site is ideally needed within 1-2 kb of the desired edit (Robert and Bessereau 2007). Zinc finger and transcription activator-like effector nucleases (Wood et al. 2011) and CRISPR/Cas9 have allowed for similar efficient editing without the constraint of transposon insertions. In particular, the ease and rapidity of generating new sgRNAs for the CRISPR/Cas9 system means that transgenic strains can be created precisely and rapidly and any endogenous NGG sequence can theoretically be targeted. Several CRISPR/ Cas9 systems have been described, each with individual strengths and weaknesses (Waaijers and Boxem 2014). Cas9 can be delivered by micro-injection of in vitro transcribed mRNA (Chiu et al. 2013; Lo et al. 2013), pure protein (Cho et al. 2013) , or plasmid DNA (Chen et al. 2013b; Dickinson et al. 2013; Friedland et al. 2013; Katic and Grosshans 2013; Waaijers étal. 2013). Similarly, the sgRNAs can be introduced by in vitro transcription, which does not require polyA tailing or 59 methyl cap addition (Chiu et al. 2013; Cho et al. 2013; Lo et al. 2013), or driven by RNA polymerase III promoters such as U6 (Chen et al. 2013b; Dickinson et al. 2013; Friedland et al. 2013; Katic and Grosshans 2013; Waaijers et al. 2013) or rpr-1 (Chiu et al. 2013). Most groups use the chimeric sgRNA, though a previous report described higher in vitro nuclease activity using the two separate bacterial small RNAs (Lo et al. 2013). Multiple groups have developed protocols for both knockouts and knock-ins. Knock-ins have been primarily generated through efficient selection schemes based on the earlier MosI-mediated single-copy transgene insertion methods using genetic markers such as unc-119 (Dickinson et al. 2013), drug resistance markers (Chen et al. 2013b), or fluorescence (Tzur et al. 2013). Typically, plasmid repair templates with 1 kb or more of homology flanking the insert have been used (Chen et al. 2013b; Dickinson et al. 2013; Tzur et al. 2013; Kim et al. 2014) .

Recently, several reports have described methods to introduce single-basepair changes, small epitopes, and larger tags such as GFP without the need for selectable markers; these approaches either directly screened all F1 progeny from co-injection marker positive animals (Paix et al. …

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