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Adam M. Andruska
  • Stanford University School of Medicine Stanford
Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
Authors:  Kabin Xie, Bastian Minkenberg and Yinong Yang, date: 09/05/2014, view: 34344, Q&A: 0
RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this protocol, we describe the general procedures for plant RGE using CRISPR-Cas9 system and Agrobacterium-mediated transformation. The protocol includes gRNA design, Cas9-gRNA plasmid construction and mutation detection (genotyping) for rice RGE and could be adapted for other plant species.
Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
Authors:  Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 26468, Q&A: 1
With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid that allows starting a new mutation after only two working days. Our protocol allows multiple, stepwise gene knockout strains with high mutagenesis efficiencies applicable for high-throughput approaches.
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