Details
| Originalsprache | Englisch |
|---|---|
| Seiten (von - bis) | 155-168 |
| Seitenumfang | 14 |
| Fachzeitschrift | Plant Journal |
| Jahrgang | 89 |
| Ausgabenummer | 1 |
| Publikationsstatus | Veröffentlicht - 14 Nov. 2016 |
| Extern publiziert | Ja |
Abstract
Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Genetik
- Agrar- und Biowissenschaften (insg.)
- Pflanzenkunde
- Biochemie, Genetik und Molekularbiologie (insg.)
- Zellbiologie
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in: Plant Journal, Jahrgang 89, Nr. 1, 14.11.2016, S. 155-168.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit
AU - Ordon, Jana
AU - Gantner, Johannes
AU - Kemna, Jan
AU - Schwalgun, Lennart
AU - Reschke, Maik
AU - Streubel, Jana
AU - Boch, Jens
AU - Stuttmann, Johannes
N1 - Funding information: We thank Sylvestre Marillonnet, Nicola Patron and Vladimir Nekrasov and Sophien Kamoun for providing the Modular Cloning Toolkit (Addgene #1000000044), the Modular Cloning Plant Parts (Addgene #1000000047) and hCas9 modules (Addgene #49771 and #49770), respectively. Christine Wagner, Hannelore Espenhahn and Samuel Grimm are acknowledged for excellent technical assistance. This work and JO and JG were funded by a grant from the Deutsche Forschungsgemeinschaft to the Collaborative Research Centre (CRC) SFB 648. MR and JStr were supported by a grant from the Deutsche Forschungsgemeinschaft (BO 1496/8-1 to JB), a grant from the European Regional Development Fund of the European Commission to JB, and by the COST action FA1208 ‘SUSTAIN’. The authors do not declare any conflict of interest.
PY - 2016/11/14
Y1 - 2016/11/14
N2 - Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.
AB - Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.
KW - Arabidopsis thaliana
KW - chromosomal deletion
KW - CRISPR/Cas
KW - EDS1
KW - Nicotiana benthamiana
KW - plant immunity
KW - technical advance
UR - http://www.scopus.com/inward/record.url?scp=85003676665&partnerID=8YFLogxK
U2 - 10.1111/tpj.13319
DO - 10.1111/tpj.13319
M3 - Article
C2 - 27579989
AN - SCOPUS:85003676665
VL - 89
SP - 155
EP - 168
JO - Plant Journal
JF - Plant Journal
SN - 0960-7412
IS - 1
ER -