Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.
The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Ting-ting
TT
Liu
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Di
D
Fan
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Ling-yu
LY
Ran
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Yuan-zhong
YZ
Jiang
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Rui
R
Liu
Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.
Ke-ming
KM
Luo
D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004252:DNA Mutational Analysis; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D016296:Mutagenesis; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D032107:Populus / Q000201:enzymology / Q000235:genetics*; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
D010940:Plant Proteins; D010088:Oxidoreductases; C022398:phytoene dehydrogenase