Academic journal article Genetics

CRISPR-Based Methods for Caenorhabditis Elegans Genome Engineering

Academic journal article Genetics

CRISPR-Based Methods for Caenorhabditis Elegans Genome Engineering

Article excerpt

Afundamental goal of biological research is to understand the functions of genes. One common strategy for studying gene function is to observe the phenotypes of mutants to deduce the biological processes in which a gene participates and, sometimes, details of its mechanism of action. This basic idea is the foundation of classical genetics and also underlies reverse genetic approaches including RNAi. A second strategy is to observe the localization and dynamics of a gene's protein product within a cell or animal, either by antibody staining or by expressing a fluorescent protein (FP) fusion. Together, these two basic strategies form the backbone of much research in Caenorhabditis elegans and other model systems.

The use of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome engineering (Hsu et al. 2014) has greatly facilitated the study of gene function in Caenorhabditis elegans and other organisms. By making precisely targeted mutations in endogenous genes, an investigator can examine the relationship between gene function and phenotype. By inserting coding sequence for a fluorescent protein, the expression and localization of endogenous proteins can be monitored. In both cases, one avoids the caveats of overexpression and silencing that are associated with conventional transgenes. Moreover, for fluorescent protein (FP) fusions, insertion of the FP into the endogenous locus allows one to use phenotypic assays to quickly determine whether the resulting fusion protein is functional. Together, these advantages permit more carefully controlled experiments to be done and thus allow greater confidence in the results. As an added benefit, current CRISPR-based approaches (Arribere et al. 2014; Dickinson et al. 2015; Paix et al. 2015; Ward 2015) are faster and require less labor than either conventional transgenesis (Mello et al. 1991) or microparticle bombardment (Praitis et al. 2001), and they eliminate the need for specialized strain backgrounds that are required for these methods and those based on the Mos1 transposon (Robert and Bessereau 2007; Frøkjaer-Jensen et al. 2008, 2010, 2012).

Many different CRISPR approaches have been developed for C. elegans and are being widely adopted by the research community. In general, all of these methods work well, with different strategies being best suited to different experimental goals. By choosing the appropriate strategy, one can now make essentially any desired change to the C. elegans genome in a matter of days to weeks, with ,1 day of hands-on labor (Dickinson et al. 2013, 2015; Arribere et al. 2014; Zhao et al. 2014; Paix et al. 2015; Ward 2015). The goal of this article is to aid users in choosing the best strategy for a given application. We provide an overview of CRISPR-based methods for C. elegans, including a discussion of which strategies are most appropriate for generating different kinds of modifications.

Overview of the CRISPR-Cas9 system

Cas9 is an endonuclease found in Archaea and some bacteria, where it is involved in adaptive immunity against phages and plasmids (Hsu et al. 2014). Unlike restriction endonucleases, whose protein structures recognize particular DNA sequences (e.g., EcoRI recognizes GAATTC), the specificity of Cas9 is determined by the sequence of an associated small RNA molecule (Figure 1) (Jinek et al. 2012). In its native context, bacterial Cas9 binds two small RNAs: a CRISPR RNA (crRNA) that determines target specificity and a trans-activating CRISPR RNA (tracrRNA) that base pairs with the crRNA and activates the Cas9 enzyme. The two RNA molecules can be fused to generate a chimeric single guide RNA (sgRNA) that supports Cas9 cleavage of DNA substrates (Jinek et al. 2012). The 20-bp guide sequence at the 59 end of the sgRNA directly determines the sequence cleaved by Cas9, by forming Watson-Crick base pairs with the DNA target (Figure 1). In addition to this base-pairing interaction, Cas9 must interact with a protospacer-adjacent motif (PAM) on the target DNA molecule. …

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