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  Editors
 Cre recombinase
 SaCas9
  Guide Target Locus
 Ai9/Ai14 reporter transgene
 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

Selection_of_gRNA_sequences_and_gRNA_scaffold_modification_lead_to_improved_editing_of_the_Ai9_locus_in_vitro_
Editing Efficiency
Record Id Condition Editor Model Target Locus Guide Vector Editing Efficiency % tdTomato+ cells Image
15000000562 Cre postive control Cre recombinase Ai9 mouse immortalized fibroblasts
15000000563 SaLoxP1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14/mTmG and related reporter transgene (within loxP) SaLoxP1-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-LoxP1
15000000564 SaLoxP1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14/mTmG and related reporter transgene (within loxP) SaLoxP1-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-LoxP1
15000000565 SaLoxP2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14/mTmG and related reporter transgene (within loxP) SaLoxP2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-LoxP2
15000000566 SaLoxP2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14/mTmG and related reporter transgene (within loxP) SaLoxP2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-LoxP2
15000000567 L1+R1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-unmodifiedR1-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L1; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R1
15000000568 L1+R1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-modifiedR1-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L1; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R1
15000000569 L1+R2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-unmodifiedR2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L1; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R2
15000000570 L1+R2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-modifiedR2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L1; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R2
15000000571 L2+R1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-unmodifiedL2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L2; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R1
15000000572 L2+R1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-modifiedL2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L2; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R1
15000000573 L2+R2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-unmodifiedL2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L2; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R2
15000000574 L2+R2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-modifiedL2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L2; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R2
15000000575 L3+R1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-unmodifiedL3-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L3; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R1
15000000576 L3+R1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-modifiedL3-modifed AAV-CMV-SaCas9-U6-modified scaffold-Sa-L3; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R1
15000000577 L3+R2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-unmodifiedL3-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L3; AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R2
15000000578 L3+R2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-modifiedL3-modifed AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L3; AAV-CMV-SaCas9-U6-modified scaffold-Sa-R2
15000000579 L1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L1
15000000580 L1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L1-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L1
15000000581 L2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L2
15000000582 L2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-L2
15000000583 L3, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L3-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-L3
15000000584 L3, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene L3-modifed AAV-CMV-SaCas9-U6-modified scaffold-Sa-L3
15000000585 R1, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R1
15000000586 R1, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R1-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-R1
15000000587 R2, unmodified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-unmodified AAV-CMV-SaCas9-U6-gRNA-LoxP-Sa-R2
15000000588 R2, modified scaffold SaCas9 Ai9 mouse immortalized fibroblasts Ai9/Ai14 reporter transgene R2-modified AAV-CMV-SaCas9-U6-modified scaffold-Sa-R2

Title Description File Download SCGE ID
Deverman_AAV Production and Administration Protocol 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. Deverman_AAV_production_and_administration_Nat Protocols-Challis_et_al_2019.pdf 21000000010
Deverman_Comprehensive Methods Procedure for plasmid cloning, editing evaluation in fibroblast, AAV production and administration, tissue processing and IHC. Deverman_Comprehensive_methods.pdf 21000000012
Deverman_Area Based Quantification of Editing Efficiency Protocol Procedure for non-IHC based image quantification of editing. Deverman_Area_based_quantification_of_editing_efficiency.pdf 21000000011

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.