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
Review
. 2023 Feb:79:102876.
doi: 10.1016/j.copbio.2022.102876. Epub 2023 Jan 6.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots: a perfect match for gene functional analysis and crop improvement

Josefa M Alamillo  1 , Cristina M López  2 , Félix J Martínez Rivas  3 , Fernando Torralbo  2 , Mustafa Bulut  3 , Saleh Alseekh  4
Affiliations
Review

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots: a perfect match for gene functional analysis and crop improvement

Josefa M Alamillo et al. Curr Opin Biotechnol. 2023 Feb.

Abstract

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene editing has become a powerful tool in genome manipulation for crop improvement. Advances in omics technologies, including genomics, transcriptomics, and metabolomics, allow the identification of causal genes that can be used to improve crops. However, the functional validation of these genetic components remains a challenge due to the lack of efficient protocols for crop engineering. Hairy roots gene editing using CRISPR/Cas, coupled with omics analyses, provide a platform for rapid, precise, and cost-effective functional analysis of genes. Here, we describe common requirements for efficient crop genome editing, focused on the transformation of recalcitrant legumes, and highlight the great opportunities that gene editing in hairy roots offers for future crop improvement.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

ga1
Graphical abstract
Figure 1
Figure 1
CRISPR/Cas gene editing and DNA repair mechanisms. Canonical Cas proteins use an RNA guide with 20 nt complementary to target DNA sequences adjacent to the 3-nt PAM (NGG in Cas9) sequence. Cas nucleases cause DSB in the DNA, which could be repaired by NHEJ or by HDR. NHEJ repair produces insertions or deletions, while HDR uses recombination with a DNA template whose ends are homologous to the break ends. This error-free DNA repair copies the DNA template from the wild-type (WT) or, from any supplied sequence, as transgene if the new sequence carries homologies at its ends. Cas variants, lacking activity of one or the two nuclease domains, can be used to directly write the genome. Created with BioRender.com.
Figure 2
Figure 2
Hairy root gene-editing workflow. Gene editing using CRISPR/Cas in plants involves different steps. Among them, the following stand out: (1) the identification of the gene to be edited, (2) the design of the gRNAs and primers for the cloning, (3) the choice of the appropriate Cas protein, (4) transformation of R. rhizogenes strain, (5) hairy root transformation, (6) sample analysis and mutation identification, and (7) interpretation of the omics and physiological changes generated in the successfully edited tissue. (cDNA, complementary deoxyribonucleic acid; Cas, cutting enzyme). Created with BioRender.com.
See this image and copyright information in PMC

References

    1. Pereira A. Plant abiotic stress challenges from the changing environment. Front Plant Sci. 2016;7:2013–2015. - PMC - PubMed
    1. Renzi J.P., Coyne C.J., Berger J., von Wettberg E., Nelson M., Ureta S., Hernández F., Smýkal P., Brus J. How could the use of crop wild relatives in breeding increase the adaptation of crops to marginal environments? Front Plant Sci. 2022;13 - PMC - PubMed
    1. Coyne C.J., Kumar S., von Wettberg E.J.B., Marques E., Berger J.D., Redden R.J., Ellis T.H.N., Brus J., Zablatzká L., Smýkal P. Potential and limits of exploitation of crop wild relatives for pea, lentil, and chickpea improvement. Legum Sci. 2020;2
    1. Fernie A.R., Yan J. De novo domestication: an alternative route toward new crops for the future. Mol Plant. 2019;12:615–631. - PubMed
    1. Zsögön A., Čermák T., Naves E.R., Notini M.M., Edel K.H., Weinl S., Freschi L., Voytas D.F., Kudla J., Peres L.E.P. De novo domestication of wild tomato using genome editing. Nat Biotechnol. 2018;36:1211–1216. - PubMed

    2. One of case studies that demonstrate CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines as a means of de novo domestication.

Publication types

Substances