TY - JOUR
T1 - High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease
AU - Eid, Ayman
AU - Ali, Zahir
AU - Mahfouz, Magdy M.
N1 - KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank Qi-Jun Chen, State Key Laboratory of Plant Physiology and Biochemistry China Agricultural University, for providing the pHEE2A-TRI vector backbone. We thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments. The study was supported by the King Abdullah University of Science and Technology.
PY - 2016/5/28
Y1 - 2016/5/28
N2 - Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.
AB - Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.
UR - http://hdl.handle.net/10754/621397
UR - http://link.springer.com/10.1007/s00299-016-2000-4
UR - http://www.scopus.com/inward/record.url?scp=84971006580&partnerID=8YFLogxK
U2 - 10.1007/s00299-016-2000-4
DO - 10.1007/s00299-016-2000-4
M3 - Article
C2 - 27236699
SN - 0721-7714
VL - 35
SP - 1555
EP - 1558
JO - Plant Cell Reports
JF - Plant Cell Reports
IS - 7
ER -