An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons. Issue 2 (10th June 2016)

• Record Type: Journal Article
• Authors: Liang, Zhen; Zong, Yuan; Gao, Caixia
• Editors: Birchler, James; Ecker, Joseph; Martin, Cathie R.; Stitt, Mark; Zhou, Jian-Min
• Other Names: Stacey Gary editorInChief.
• Is Part Of: Current protocols in plant biology. Volume 1:Issue 2(2016)
• Page Start: 329
• Page End: 344
• Publication Date: 2016-06-10
• Subjects: CRISPR/Cas -- gene knock‐out -- maize -- rice -- wheat; Botany -- Laboratory manuals; Plants; Botany; Botany; Laboratory Manual; Periodical; Laboratory manuals; 580.28
• DOI: 10.1002/cppb.20021
• Languages: English
• ISSNs: 2379-8068

Abstract

Abstract: Precise genome modification using artificial nucleases is a powerful tool for in‐depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double‐strand breaks (DSBs) into pre‐selected genomic loci and lead to loss of gene function due to error‐prone non‐homologous end joining (NHEJ). RNA‐guided nucleases have been widely used in several eukaryotic organisms. In this article, we provide a detailed protocol for designing and constructing gRNA targets, detecting nuclease activity in transient protoplast assays, and identifying mutations in transgenic plants (including rice, wheat and maize). Targeted mutations in T0 plants can be generated in 14 to 18 weeks. © 2016 by John Wiley & Sons, Inc.