Development System Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro | SCGE Toolkit Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts
Experiment: Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro
PI:
Benjamin E Deverman, PhD
Description: Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts
Editing Assay:
Editing efficiency was assessed by flow cytometry for tdTomato expression in Ai9 mouse embryonic fibroblasts after transfection with a mixture of plasmids expressing SaCas9 and the indicated U6-gRNAs
Editing efficiency was assessed by flow cytometry for tdTomato expression in Ai9 mouse embryonic fibroblasts after transfection with Cre recombinase positive control
Editing efficiency was assessed by flow cytometry for tdTomato expression in Ai9 mouse embryonic fibroblasts after transfection with an individual plasmid expressing SaCas9 and the indicated U6-gRNA
Ai9/Ai14/mTmG and related reporter transgene (within loxP)
Guides
L1-modified
L1-unmodified
L2-modified
L2-unmodified
L3-modifed
L3-unmodified
R1-modified
R1-unmodified
R2-modified
R2-unmodified
SaLoxP1-modified
SaLoxP1-unmodified
SaLoxP2-modified
SaLoxP2-unmodified
Models
Ai9 mouse immortalized fibroblasts
Vectors
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L1
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L2
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L3
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-LoxP1
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-LoxP2
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R1
AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R2
AAV-CMV-SaCas9-U6-modified scaffold-Sa-L1
AAV-CMV-SaCas9-U6-modified scaffold-Sa-L2
AAV-CMV-SaCas9-U6-modified scaffold-Sa-L3
AAV-CMV-SaCas9-U6-modified scaffold-Sa-LoxP1
AAV-CMV-SaCas9-U6-modified scaffold-Sa-LoxP2
AAV-CMV-SaCas9-U6-modified scaffold-Sa-R1
AAV-CMV-SaCas9-U6-modified scaffold-Sa-R2
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Results
SCGE Toolkit downloaded on: 2024/11/11 00:48:29; Please cite the Somatic Cell Genome Editing Consortium Toolkit NIH HG010423 when using publicly accessible data in formal presentation or publication. SCGE Experment ID: 18000000022. PI:
Benjamin E Deverman PhD
Published procedures for AAV production, delivery, and tissue imaging.
Challis et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3.
Improved editing of the Ai9 locus in vitro after optimization of gRNAs and modification of the gRNA scaffold
Figure 1. Improved editing of the Ai9 locus in vitro after optimization of gRNAs and modification of the gRNA scaffold. A. The schematic shows the Ai9 lox-stop-lox-tdTomato cassette and the positions of tested gRNA target sequences. B. gRNA sequences, PAMs and their predicted efficiency and on- and off-target editing ranks as predicted by the Broad Genetic Perturbation Platform https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design. Sequences that deviate from the NNGRRT consensus SaCas9 PAM are highlighted in red. C. A schematic of the Cas9-gRNA construct design. Modifications to the gRNA scaffold are highlighted in red. The position of the spacer is shown in blue. D. In vitro editing efficiency by SaCas9 with single or paired gRNA combinations. Ai9 fibroblasts were transfected with a single or pair of CMV-SaCas9 vectors with a U6-driven gRNA as indicated. The cells were then assessed for tdTomato expression as a marker of editing by flow cytometry. Plasmids harboring modifications to the gRNA scaffold enhance the editing of the Ai9 loxstop-lox cassette. E. Editing efficiency normalized to the recombination efficiency achieved by EF1a-Cre expression, which served as a positive control.