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Addgene

pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) Pooled Library
(Pooled Library #198050)

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  • Purpose

    This library contains approximately 81,000 unique plasmids representing all possible single codon mutations to the AAV2 rep gene, including wild type and premature stop codon controls. The pCMV-Rep78/68 SSM library enables expression of only the larger Rep proteins (Rep78/68) and is provided in an ITR backbone to enable packaging of rep variant sequences into AAV capsids. By supplying an AAV cap gene of interest in trans, researchers can use this library to investigate the effects of rep mutations on production of a wide range of AAV capsid serotypes.

  • Vector Backbone

    pCMV-Rep78-68 library backbone (GB, 16 KB)

Ordering

Item Catalog # Description Quantity Price (USD)
Pooled Library 198050 SSM library† 1 $ 640 Add to Cart
Available to Academic and Nonprofits Only

  • † A AAV-Cap is NOT included with this library and will have to be ordered separately. Recommended plasmids are pCMV-AAV2cap (#198014), pCMV-AAV5cap (#198015), and pCMV-AAV9cap (#198016).

Library Details

  • Uniquely barcoded variants
    80,962

Library Shipping

This library is delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.

  • Volume
    ∼20 µL
  • Concentration
    50 ng/µL

Resource Information

Depositor Comments

Deconvolution algorithms for analyzing NGS data: https://github.com/churchlab/aav_rep_scan (Link opens in a new window).

The barcode and NGS primer binding sites in the library constructs are added during the cloning process so are not included in the vector backbone map. The example of a final cloned product vector map (GB, 21 KB) contains the exact location of the barcode and NGS primer binding sites.


The figure has two parts, A and B. Part A is a schematic showing DNA at the top, mRNA in the middle, and protein at the bottom. Between the left and right ITRs of the linear DNA are shown 5 features: CMV, rep, BC, WPRE, and poly(A). Lined up underneath the rep/BC features (in light color) are the unspliced and spliced mRNAs (in medium color) one on top of the other. Below is the Rep78 protein and at the bottom is the Rep68 protein (in dark color) with OBD (left), HD (middle), and ZFD (right) shown. Part B depicts a schematic of the library production assay. pHelper, WT rep52/40, and AAV2 cap plasmids are transfected into HEK293 cells (shown as a flask) containing the rep78/68 Library (shown as a plasmid). Cells are Iodixanol Gradient Ultracentrifuged (shown as a centrifugation tube with bands of color) and encapsidated genomes (shown as hexagonal viral particles, 4 of the 6 containing the dark-colored DNA) are separated. The Library (f sub plasmid) and Virus (f sub virus) are barcode amplified and sequenced with arrows pointing up towards these formulas: S equals F sub virus divided by F sub plasmid and s primed equals S divided by s wild type.
  • Figure 1. pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) library design and use in production assay. (A) The pCMV-Rep78/68 SSM library contains all possible single codon deletions and substitutions between the Rep78/68 start codon and the Rep78 stop codon (dotted line). The Rep52/40 start codon was mutated to prevent expression of the smaller Rep proteins and enable investigation of only the larger Rep proteins. (B) Overview of the library production assay. Barcode amplification and sequencing from plasmid and viral pools enables measurement of the effect of all single codon rep mutations on AAV production. Any cap gene of interest can be used in place of the AAV2 cap.
How to cite this pooled library ( Back to top )

These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) library was a gift from George Church (Addgene #198050)

  • For your References section:

    Comprehensive mutagenesis maps the effect of all single codon mutations in the AAV2 rep gene on AAV production. Jain NK, Ogden PJ, Church GM bioRxiv 2023.01.31.526541. doi: 10.1101/2023.01.31.526541. bioRxiv 2023.01.31.526541.
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