摘要
The newly developed CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations andcorrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA(sg RNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sg RNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.
The newly developed CRISPR （Clustered Regularly Interspaced Short Palindromic Repeats）/Cas （CRISPR-associated） system has emerged as an efficient tool for genome-editing, providing an alternative to clas- sical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that intro- duce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations and corrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA （sgRNA）, vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sgRNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.
基金
supported by the Chinese Academy of Sciences and China Scholarship Council(201206050103)
