The pAAV.CMV.Luc.IRES.EGFP.SV40, dCas9-VPR, pSMVP-Cas9N, pSMVP-Cas9C, pAAV-CMV-Cas9C-VPR, and pCMV-PE2 plasmids were obtained from Addgene (#105533, #63798, #80934, #80939, and #80933, #132775). REVeRT-based reporter genes, (d)Cas9-VPR, PE2 and ABCA4 constructs were synthesized (BioCat GmbH) and cloned into an ITR-containing plasmid (pAAV2.1). The luciferase constructs were generated using the pAAV.CMV.Luc.IRES.EGFP.SV40 plasmid and cloned into pAAV2.1. Binding domains and splice sites were exchanged, promoter and pA signal removed and sgRNA expression cassettes added32 (link) using standard cloning techniques. All constructs were sequenced before use (Eurofins Genomics).
Psmvp cas9n
Manufactured by Addgene
PSMVP-Cas9N is a Cas9 variant that has been engineered to have reduced nuclease activity, making it a 'nickase' enzyme. It can be used for targeted genome editing applications that require single-strand DNA breaks rather than double-strand breaks.
Automatically generated - may contain errors
2 protocols using psmvp cas9n
1
Versatile Plasmid Toolkit for Gene Editing
2
Inducible and Tissue-Specific CRISPR-Cas9 Activation
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The Cas9m4-VP64, SP-dCas9-VPR, pSMVP-Cas9N, pSMVP-Cas9C, and pAAV-CMV-Cas9C-VPR plasmids were obtained from Addgene (#47319, #63798, #80934, #80939, and #80933, respectively). sgRNAs expressed via a U6 promoter were added using standard cloning techniques. Cas9N and Cas9C were rendered catalytically inactive by introducing the D10A and the H840A point mutations via a standard site-directed mutagenesis protocol using the KAPA HiFi HotStart ReadyMix PCR kit (Kapa Biosystems). For the generation of stable cell lines, dCas9-VPR was subcloned into a pb expression vector containing the Tet-On system for DOX-inducible expression of dCas9-VPR. For expression in mouse photoreceptors, the split dCas9-VPR driven by a human rhodopsin promoter and corresponding sgRNAs each driven by a human U6 promoter were subcloned into the pAAV2.1 vector (27 (link)). All transgenes were sequenced before use (Eurofins Genomics).
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