A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

Frontiers in Plant Science
Karolina HusRobert Hasterok

Abstract

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temper...Continue Reading

References

Oct 27, 2004·Plant Cell Reports·Pernille ChristiansenKlaus Kristian Nielsen
Jul 3, 2007·Nature Biotechnology·Jeffrey C MillerEdward J Rebar
May 29, 2010·Proceedings of the National Academy of Sciences of the United States of America·Feng ZhangDaniel F Voytas
Jul 28, 2010·Genetics·Michelle ChristianDaniel F Voytas
Dec 15, 2010·Nature Methods·Jeffry D SanderJ Keith Joung
Dec 24, 2010·Nature Biotechnology·Jeffrey C MillerEdward J Rebar
May 9, 2012·Nature Biotechnology·Ting LiBing Yang
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 5, 2013·Molecular Plant·Qiwei ShanCaixia Gao
Aug 21, 2013·Molecular Plant·Kabin Xie, Yinong Yang
Sep 4, 2013·Cell Research·Jin MiaoLi-Jia Qu
Oct 15, 2013·G3 : Genes - Genomes - Genetics·Santosh Kumar UpadhyayRakesh Tuli
Jan 29, 2014·Proceedings of the National Academy of Sciences of the United States of America·Tao ZhengJohn H Doonan
Sep 19, 2014·Nature Protocols·Qiwei ShanCaixia Gao
Mar 12, 2015·Methods in Molecular Biology·Ha-il JungOlena K Vatamaniuk
Dec 21, 2016·Plant Cell Reports·Shouwei TianYong Xu
Apr 23, 2017·The Plant Journal : for Cell and Molecular Biology·Mon Mandy HsiaJohn P Vogel
Oct 25, 2017·ELife·Devin Lee O'ConnorOttoline Leyser
Jan 9, 2018·Biochemical and Biophysical Research Communications·Jingwei YanAying Zhang
Jun 28, 2018·The New Phytologist·Alja van der SchurenChristian S Hardtke
Feb 26, 2019·Frontiers in Plant Science·Satya Swathi NadakudutiDavid S Douches
May 28, 2019·International Journal of Molecular Sciences·Artur PinskiRobert Hasterok
Jul 28, 2019·International Journal of Molecular Sciences·Tobias BruegmannMatthias Fladung

❮ Previous
Next ❯

Citations

Jul 3, 2021·International Journal of Molecular Sciences·Artur PinskiRobert Hasterok
Jul 25, 2021·International Journal of Molecular Sciences·Artur PinskiRobert Hasterok

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
Electrophoresis
Assay
transfection
transgenic
restriction

Software Mentioned

Cas
OFFinder
Geneious Prime

Related Concepts

Related Feeds

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

Cell Checkpoints & Regulators

Cell cycle checkpoints are a series of complex checkpoint mechanisms that detect DNA abnormalities and ensure that DNA replication and repair are complete before cell division. They are primarily regulated by cyclins, cyclin-dependent kinases, and the anaphase-promoting complex/cyclosome. Here is the latest research.