CRISPR/Cas9-mediated genome editing technology has been used to successfully edit numerous genes in various organisms including plants. There are still two major challenges in using CRISPR/Cas9 technology for gene editing in plants. First, there are very limited choices of promoters that are suitable for in vivo production of single-guide RNAs (sgRNAs), which is complementary to the target sequence and which guides Cas9 to generate double-strand breaks at the target site. It is especially difficult to produce sgRNA molecules with temporal and spatial precision. Second, there is a lack of efficient methods for identifying plants that (1) contain heritable and stable mutations generated by CRISPR/Cas9, and (2) no longer harbor the CRISPR/Cas9 construct and other transgenes. In this chapter, we describe the development of a ribozyme-based strategy that enables the production of sgRNA molecules from any chosen promoter. More importantly, the ribozyme-based technology makes it feasible to produce sgRNAs with temporal and spatial precision, greatly expanding the scope and applications of CRISPR/Cas9 technology. We also developed a fluorescence-based technology that allows us to efficiently and reliably isolate Cas9-free stable Arabidopsis mutants. Thus, we provide effective protocols to overcome two important obstacles in using CRISPR/Cas9 for editing genes in plants.
Keywords: Arabidopsis; CRISPR; Cas9; RGR; Ribozyme; mCherry.
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