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Rice Gene Knockout or Downregulation through CRISPR-Cas9

  • Yvonne Ludwig
  • Inez H. Slamet-LoedinEmail author
Protocol
  • 23 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Precise genome modification via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9 in rice plants, is a powerful tool for improving abiotic and biotic stress tolerance and increasing yield in rice. Valuable agronomical traits and functional study of a novel gene can be obtained by targeting single or multiple mutations in the rice genome. Here we describe the sgRNA design, cloning, and assembly of CRISPR constructs for specific gene knockout and the variant analysis in the rice plant.

Key words

CRISPR-Cas9 sgRNA design Knockout Mutagenesis Rice 

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

BAP

6-Benzylaminopurine

Cas9

CRISPR-associated protein 9

CRISPR

Clustered regularly interspaced short palindromic repeats

dATP

Deoxyadenosine triphosphate

DNA

Deoxyribonucleic acid

GMO

Genetically modified organism

HPT

Hygromycin phosphotransferase gene

IE

Immature embryo

Indels

Insertions and deletions

LB

Luria broth

MES

2-(N-Morpholino)ethanesulfonic acid

MS medium

Murashige and Skoog medium

NAA

1-Naphthaleneacetic acid.

PAM

Protospacer adjacent motif

PCR

Polymerase chain reaction

pDNA

Plasmid DNA

PNK

Polynucleotide kinase

RT

Room temperature

sgRNA

Single-guide ribonucleic acid

SOC medium

Super optimal broth

T7E1

T7 Endonuclease 1

YEP

Yeast extract peptone dextrose

Notes

Acknowledgments

We want to thank Daniel Voytas Laboratory for providing us the modular vector system.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Strategic Innovation PlatformInternational Rice Research InstituteLos BanosPhilippines

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