Benefits Of Using The PrecisionX™ Multiplex gRNA Cloning Kits
• Extremely efficient cloning with a single kit
• Compatible with most Cas9/cloning vectors
How the PrecisionX™ Multiplex gRNA Cloning Kit Works
The PrecisionX multiplex gRNA cloning kit provides the H1 and U6 promoter blocks to easily build multiple gRNA cassettes. In step 1, primers with overlapping ends containing the the desired guide RNA (designed by user) and the scaffold-promoter block (from the kit) are combined in a PCR reaction to generate the PCR amplicon containing both gRNAs. Secondly, the PCR amplicon created in step 1 is combined with the fusion reaction mix along with the linearized destination expression vector for seamless construction. The cloning process is extremely efficient and the kit can be adapted to two or more gRNAs in a single fusion reaction.
Dual gRNA Cloning & KO Data
A dual gRNA CRISPR/Cas9 vector with two separate gRNAs targeting a transgenic reporter HEK293 cell stably expressing an RFP reporter gene were designed. The dual gRNAs targeting RFP (gRNA1 and gRNA7) were fused into SBI's CAS940A-1 vector (CMV-hspCas9-H1-gRNA) using the Multiplex gRNA Cloning Kit. PCR assays using primers flanking the RFP gene were utilized to assess the cutting efficiencies of the dual gRNA CRISPR/Cas9 vector. If no gRNA-directed cutting occured, a 711 bp band is observed. If dual cutting occurs, then a 458 bp piece is cut out of the RFP gene and a resulting 253 bp PCR product is generated. The results from the dual gRNA1 plus gRNA7 treated and untreated reporter cells indicate evidence of cutting by the dual gRNAs, and fluorescence images of targeted cells vs control show reduction in RFP expression (data not shown).
Material Available For Download
PrecisionX Multiplex gRNA Cloning Kit User Manual
PrecisionX Multiplex gRNA with Cas9 Vector Combo Kits User Manual
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