Tissue-specific gene targeting using CRISPR/Cas9

Abstract

The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.

Keywords: CRISPR/Cas9 technology; Gene knockout; Morpholino; Tissue specificity; gRNAs.

FIGURE 1

CRISPR sequences against any gene of interest can be identified in silico through published algorithms. gRNAs can then be prepared by in vitro transcription (using T7 or SP6 RNA polymerases) from DNA templates comprising an RNA polymerase promoter, the gene-specific seed sequence, and the sequence encoding the constant 3′ part of the gRNA. Purified gRNAs can be injected into single-cell zebrafish embryos along with Cas9 mRNA or protein. Finally, genomic DNA can be extracted from injected embryos and analyzed for CRISPR-induced mutations, for example, by direct sequencing, HRM, or T7E1 mutagenesis assay.

FIGURE 2

A Gateway reaction allows to assemble a Cas9 sequence under the control of a tissue-specific promoter and followed by polyA into a backbone containing a U6:gRNA cassette and possibly a transgenesis marker. One can then clone any gene-specific seed sequence into the U6:gRNA cassette of the resulting vector. This tissue-specific CRISPR vector can be injected with Tol2 mRNA into one cell–stage embryos that will later be submitted to phenotype evaluation. (See color plate)