Molecular Cloning Techniques for AAV Production

Standard molecular cloning techniques were used to generate DNA constructs in this study. Double-stranded DNA was synthesized by Integrated DNA Technologies and inserted into pAAV backbones with NEBuilder HIFI (New England Biolabs, E2621). sgRNA sequences were synthesized as overlapping single-stranded DNA oligos (Integrated DNA Technologies) that were then annealed together and ligated into sgRNA expression cassettes using T4 DNA ligase (New England Biolabs, M0202).
pUCmini-iCAP-AAV-PHP.eB^{12 (link)} (Addgene #175004), pUCmini-iCAP-AAV.CAP-B10^{14 (link)} (Addgene #103005), pUCmini-iCAP-AAV.MaCPNS2^{15 (link)} (Addgene #185137), AAV-DJ rep-cap (Cell Biolabs, VPK-420-DJ), AAV6 rep-cap (Cell Biolabs, VPK-420-DJ), and pHelper (Agilent, #240071) plasmids were used for production of AAVs. Prior to use, all plasmids were sequence verified via whole-plasmid sequencing (Primordium Labs).

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Coughlin G.M., Borsos M., Appling N., Barcelona B.H., Mayfield A.M., Mackey E.D., Eser R.A., Chen X., Kumar S.R, & Gradinaru V. (2023). Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo. bioRxiv.

Publication Preprint 2023

NEBuilder HIFI T4 DNA ligase VPK-420-DJ pHelper

Corresponding Organization :

Other organizations : California Institute of Technology

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Variable analysis

independent variables

Molecular cloning techniques used to generate DNA constructs
SgRNA sequences synthesized as overlapping single-stranded DNA oligos

dependent variables

Production of AAVs

control variables

Use of pUCmini-iCAP-AAV-PHP.eB, pUCmini-iCAP-AAV.CAP-B10, and pUCmini-iCAP-AAV.MaCPNS2 plasmids
Use of AAV-DJ rep-cap, AAV6 rep-cap, and pHelper plasmids
Sequence verification of all plasmids via whole-plasmid sequencing

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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