{U6-(gRNA)}*3-hSyn-mCherry-KASH-hGHpA

Triple-gRNA vector expressing three independent gRNAs from tandem U6 promoters alongside hSyn-driven mCherry-KASH for FANS-compatible nuclear envelope labeling in neurons. Directly adapted from Kim et al. 2021 (Sci Rep, doi:10.1038/s41598-021-82287-0) CSAC design (pAAV-(hU6-gRNA)x3-hSyn-mCherry-KASH) with hGHpA as terminator. No Addgene deposit exists for the Kim 2021 plasmids.

Characteristics

Triple U6-gRNA cassettes assembled via Golden Gate cloning enable simultaneous targeting of three genomic loci. hSyn-driven mCherry-KASH restricts reporter to neurons and anchors mCherry to the outer nuclear membrane for FANS. Red channel is compatible with EGFP-based co-reporters or imaging probes. Validated in the Kim 2021 CSAC system for multiplexed clock gene knockout in mouse SCN.

Parts

mCherry KASH Domain hSyn Promoter hGH PolyA Signal U6 Promoter

Custom Inserts

SpCas9 gRNA target (20-23 bp) SpCas9 gRNA target (20-23 bp) SpCas9 gRNA target (20-23 bp)

Related Designs

U6-(gRNA)-hSyn-EGFP-KASH-hGHpA {U6-(gRNA)}*3-hSyn-mCherry-KASH-hGHpA (pro)-SpCas9-SV40pA + 3*{U6-(gRNA)}-hSyn-mCherry-KASH-bGHpA (pro)-SpCas9-SV40pA + U6-(gRNA)-hSyn-mCherry-bGHpA (pro)-SpCas9-SV40pA + 2*{U6-(gRNA)}-CAG-mCherry-bGHpA

Literature Sources

  1. Kim et al. (2021). Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes. Sci Rep- Kim 2021 Multiplex AAV

Available Products

No products currently available with this design. Contact us for custom orders.

Additional Information

Validated in: Neuro2a cells; mouse suprachiasmatic nucleus (SCN) in vivo, AAV-DJ serotype; organotypic SCN slice cultures

Applications: Multiplex 3-target neuronal CRISPR editing in vivo for redundant gene families (e.g., Cry1/Cry2, Per1/Per2) where single knockouts are insufficient. mCherry-KASH enables FANS-based isolation of transduced neuronal nuclei and immunohistochemical verification of transduction. Requires co-delivery with a Cas9-expressing vector.

Images