An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing
- PMID: 27208253
- PMCID: PMC4936589
- DOI: 10.1104/pp.16.00663
An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing
Abstract
Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.
© 2016 American Society of Plant Biologists. All Rights Reserved.
Figures
Similar articles
-
Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.Methods Mol Biol. 2019;1917:147-154. doi: 10.1007/978-1-4939-8991-1_11. Methods Mol Biol. 2019. PMID: 30610634
-
Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles.Planta. 2015 Jan;241(1):271-84. doi: 10.1007/s00425-014-2180-5. Epub 2014 Oct 1. Planta. 2015. PMID: 25269397 Free PMC article.
-
A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant. 2015 Aug;8(8):1274-84. doi: 10.1016/j.molp.2015.04.007. Epub 2015 Apr 24. Mol Plant. 2015. PMID: 25917172
-
On Improving CRISPR for Editing Plant Genes: Ribozyme-Mediated Guide RNA Production and Fluorescence-Based Technology for Isolating Transgene-Free Mutants Generated by CRISPR.Prog Mol Biol Transl Sci. 2017;149:151-166. doi: 10.1016/bs.pmbts.2017.03.012. Epub 2017 May 3. Prog Mol Biol Transl Sci. 2017. PMID: 28712495 Review.
-
[CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants].Yi Chuan. 2016 Feb;38(2):118-25. doi: 10.16288/j.yczz.15-395. Yi Chuan. 2016. PMID: 26907775 Review. Chinese.
Cited by 45 articles
-
Interaction Between AtCML9 and AtMLO10 Regulates Pollen Tube Development and Seed Setting.Front Plant Sci. 2020 Jul 23;11:1119. doi: 10.3389/fpls.2020.01119. eCollection 2020. Front Plant Sci. 2020. PMID: 32793269 Free PMC article.
-
Easy-to-Use InDel Markers for Genetic Mapping between Col-0 and Ler-0 Accessions of Arabidopsis thaliana.Plants (Basel). 2020 Jun 22;9(6):779. doi: 10.3390/plants9060779. Plants (Basel). 2020. PMID: 32580428 Free PMC article.
-
A Simple and Fast Manual Centrifuge to Spin Solutions in 96-Well PCR Plates.Methods Protoc. 2020 May 25;3(2):41. doi: 10.3390/mps3020041. Methods Protoc. 2020. PMID: 32466310 Free PMC article.
-
Plant Genome Editing and the Relevance of Off-Target Changes.Plant Physiol. 2020 Aug;183(4):1453-1471. doi: 10.1104/pp.19.01194. Epub 2020 May 26. Plant Physiol. 2020. PMID: 32457089 Free PMC article.
-
Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.BMC Plant Biol. 2020 May 25;20(1):234. doi: 10.1186/s12870-020-02385-5. BMC Plant Biol. 2020. PMID: 32450802 Free PMC article. Review.
Publication types
MeSH terms
Substances
Grant support
LinkOut - more resources
-
Full Text Sources
-
Other Literature Sources
-
Molecular Biology Databases
-
Research Materials
-
Miscellaneous