CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice
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CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants.
The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.
KeywordsCRISPR Cas9 Cpf1 Stomata Rice OsEPFL9
We thank Prof. Caixia Gao of Chinese Academy of Science, China for providing us Cas9 and gRNA scaffold constructs. Also we would like to thank Dr. Feng Zhang of Broad Institute MIT for providing us pcDNA3-huLbCpf1. We also thank Florencia Montecillo and Juvy Reyes for helping in rice transformation and molecular work respectively.
Author contribution statement
XY, AKB and JD contributed equally. AB, PQ, JG and XY designed the experiments and wrote the manuscript. Vector designing and construction was done by XY, AKB, AB and TK (CRISPR-508 Cas9). XY,SM, CPB, CC, AB (CRISPR-Cpf1). Surveyor assay and sequencing was performed 509 by KP, XY, SM, CPB (Cas9 plants and Cpf1 plants) and AKB (Cas9 plants). Rice 510 transformation was supervised by XY. Microscopy was performed by JD, HL and RC. Data 511 analysis was performed by AB and XY. Southern blotting was performed by CPB.
AB, PQ, JG and XY designed the experiments and wrote the manuscript. Vector designing and construction was done by XY, AKB, AB and TK (CRISPR-Cas9). XY,SM, CPB, CC, AB (CRISPR-Cpf1). Surveyor assay and sequencing was performed by KP, XY, SM, CPB (Cas9 plants and Cpf1 plants) and AKB (Cas9 plants). Rice transformation was supervised by XY. Microscopy was performed by JD, HL and RC. Data analysis was performed by AB and XY. Southern blotting was performed by CPB.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interests.
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