TY  - BOOK
AU  - He,Y.
AU  - Wang,R.
AU  - Dai,X.
AU  - Zhao,Y.
TI  - On Improving CRISPR for Editing Plant Genes: Ribozyme-Mediated Guide RNA Production and Fluorescence-Based Technology for Isolating Transgene-Free Mutants Generated by CRISPR
KW  - ARABIDOPSIS
KW  - CAS9
KW  - CRISPR
KW  - MCHERRY
KW  - RGR
KW  - RIBOZYME
N2  - 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
UR  - https://drive.google.com/file/d/1rpAuNa34agjO6ut02kQcxYMIN_ejwXZ4/view?usp=drivesdk
ER  -