Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Mar;1(3):e65.
doi: 10.1002/cpz1.65.

CRISPR-Cas9 Based Genome Editing in Wheat

Mark A Smedley  1 , Sadiye Hayta  1 , Martha Clarke  1 , Wendy A Harwood  1
Affiliations

CRISPR-Cas9 Based Genome Editing in Wheat

Mark A Smedley et al. Curr Protoc. 2021 Mar.

Erratum in

Abstract

The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.

Keywords: CRISPR; Cas9; Golden Gate assembly; genome editing; knockout; wheat.

PubMed Disclaimer

References

Literature Cited

    1. Abe, F., Haque, E., Hisano, H., Tanaka, T., Kamiya, Y., Mikami, M., … Sato, K. (2019). Genome-edited triple-recessive mutation alters seed dormancy in wheat. Cell Reports, 28(5), 1362-1369.e1364. doi: 10.1016/j.celrep.2019.06.090.
    1. Adamski, N. M., Borrill, P., Brinton, J., Harrington, S. A., Marchal, C., Bentley, A. R., … Uauy, C. (2020). A roadmap for gene functional characterisation in crops with large genomes: Lessons from polyploid wheat. eLife, 9, e55646. doi: 10.7554/eLife.55646.
    1. Ansari, W. A., Chandanshive, S. U., Bhatt, V., Nadaf, A. B., Vats, S., Katara, J. L., … Deshmukh, R. (2020). Genome editing in cereals: Approaches, applications and challenges. International Journal of Molecular Sciences, 21(11), 4040. doi: 10.3390/ijms21114040.
    1. Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., … Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157. doi: 10.1038/s41586-019-1711-4.
    1. Bahassi, E. M., & Stambrook, P. J. (2014). Next-generation sequencing technologies: Breaking the sound barrier of human genetics. Mutagenesis, 29(5), 303-310. doi: 10.1093/mutage/geu031.

MeSH terms

LinkOut - more resources