Ribonucleoprotein (RNP)-Mediated Allele Replacement in Barley (Hordeum vulgare L.) Leaves
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Biolistic, CRISPR/Cas, Cereals, Homology-directed repair, Tissue culture, Triticeae,
- MeSH
- Alleles MeSH
- CRISPR-Cas Systems MeSH
- DNA MeSH
- Hordeum * genetics metabolism MeSH
- Ribonucleoproteins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA MeSH
- Ribonucleoproteins MeSH
Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.
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Rajaraman J, Douchkov D, Lück S et al (2018) Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance. Genome Biol 19:116. https://doi.org/10.1186/s13059-018-1472-7 PubMed DOI PMC
Panchy N, Lehti-Shiu M, Shiu S-H (2016) Evolution of gene duplication in plants. Plant Physiol 171:2294–2316. https://doi.org/10.1104/pp.16.00523 PubMed DOI PMC
Krattinger SG, Keller B (2016) Molecular genetics and evolution of disease resistance in cereals. New Phytol 212:320–332. https://doi.org/10.1111/nph.14097 PubMed DOI
Chauhan H, Boni R, Bucher R et al (2015) The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley. Plant J 84:202–215. https://doi.org/10.1111/tpj.13001 PubMed DOI
Wachter RM, Elsliger M-A, Kallio K et al (1998) Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6:1267–1277. https://doi.org/10.1016/S0969-2126(98)00127-0 PubMed DOI
Budhagatapalli N, Hensel G (2022) Multiplexed genome editing in plants using CRISPR/Cas-based endonuclease systems. In: Wani SH, Hensel G (eds) Genome editing. Springer, Cham, pp 143–169 DOI
Zhang Y, Iaffaldano B, Qi Y (2021) CRISPR ribonucleoprotein-mediated genetic engineering in plants. Plant Commun 2:100168. https://doi.org/10.1016/j.xplc.2021.100168 PubMed DOI PMC
Svitashev S, Schwartz C, Lenderts B et al (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:13274. https://doi.org/10.1038/ncomms13274 PubMed DOI PMC
Budhagatapalli N, Rutten T, Gurushidze M et al (2015) Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double-strand breaks in barley. G3 (Bethesda) 5:1857–1863. https://doi.org/10.1534/g3.115.018762 PubMed DOI
Budhagatapalli N, Schedel S, Gurushidze M et al (2016) A simple test for the cleavage activity of customized endonucleases in plants. Plant Methods 12:18. https://doi.org/10.1186/s13007-016-0118-6 PubMed DOI PMC
