Fig. 1.

Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene (CmR) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [22] using the In-Fusion^® HD cloning strategy as described in “Methods” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion^® HD cloning system. c pUC57GW: this is an in-house constructed Gateway^®-compatible Entry vector, which, in contrast to commonly used Gateway^® Entry/DONR vectors, contains the ccdB and Chloranphenicol (CmR) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion^® HD cloning system without any background colonies. Please see “Methods” and “Results” section for details