Maize CRISPR/CAS9 for Mutagenesis

01 November 2017 - By: Jim Ruddock

Maize CRISPR/CAS9 for Mutagenesis

An Agrobacterium-delivered CRISPR/ Cas9 system for high-frequency targeted mutagenesis in maize

 

Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, Becraft PW, Meyers BC, Walbot V, Wang K, Yang B (2016). Plant Biotechnology Journal, doi: 10.1111/pbi.12611.

Maize genome editing using CRISPR/Cas9 has previously been reported at both high and low efficiency, but with limited access to publicly available systems. In this article Char et al. describe the ISU Maize CRISPR platform, a public sector platform for maize genome editing, which is highly efficient with >70% transgenic plants carrying targeted mutations. The system consists of an Agrobacterium-delivered CRISPR/Cas system, and the manuscript describes an Agrobacterium T-DNA plasmid design and associated results using this vector for targeted mutagenesis in maize when delivering Cas9 and guide RNAs. The vector is amenable for the cloning of up to four guide RNAs for multiplex targeting of genes in maize. The authors use this vectoring system to target two genes which are duplicated, Argonaute 18 and dihydroflavonol 4-reductase (DHFR), and report high frequencies of mutagenesis. The experiments direct mutagenesis using twoguide RNAs in a single T-DNA to target each duplicate gene independently to maximize the probability of directing non-homologous end joining (NHEJ) mutations at two different sites in each allele. The authors then combine paired guide RNAs (4 guide RNAs in total) to simultaneously mutagenize the duplicated genes (two Argonaute or two DHFR genes). The authors go on to examine a limited number of progeny plants for inheritance of the mutations. When wildtype, unmutated genes are introduced through crossing with plants that contained Cas9-guide RNA T-DNA insertions, new mutations are identified which support the idea that Cas9-guide RNA remain active and are responsible for the generation of these newly identified mutations in progeny plants. The authors then outline a strategy to reduce the number of embryos per mutagenesis experiment by co-infecting with two different Agrobacterium strains. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool for targeted mutagenesis in this important crop plant.

By Jim Ruddock, Managing Editor






































Category: Plant Biology
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Jim Ruddock - Author Profile

Jim Ruddock

Jim Ruddock is the Editorial Manager for The Plant Journal and Plant Biotechnology Journal, and manages all processes of each journal through the peer review system – from submission to acceptance and export of final files to the Production department. Currently at Wiley, Jim has 23 year’s of experience working in science journal publishing environments in various capacities.