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pRep-Cap Scanning Saturation Mutagenesis (SSM) Pooled Library
(Pooled Library #199601)

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  • Purpose

    This library contains approximately 200,000 unique plasmids representing all possible single codon substitutions, deletions, and insertions in the AAV2 cap gene, including wild-type and premature stop codon controls. The pRep-Cap SSM library enables expression of VP1, VP2, and VP3 under the control of the endogenous p40 promoter, and enables in cis expression of the AAV2 rep gene. The library is provided in an inverted terminal repeat (ITR) backbone to enable packaging of cap variant sequences into AAV capsids. Researchers can use this library to investigate the effects of single codon cap mutations on AAV production and on capsid properties, such as thermostability, antibody neutralization, and transduction.

  • Vector Backbone

    pRep-Cap library backbone (GB, 14 KB)

Ordering

Item Catalog # Description Quantity Price (USD)
Pooled Library 199601 pRep-Cap SSM Library† 1 $850 Add to Cart
Available to Academic and Nonprofits Only

  • †An adenoviral helper plasmid, pAdDeltaF6 from the Wilson lab, is required for production (#112867) and will have to be ordered separately.

Library Details

  • Vector type
    Mammalian Expression, AAV
  • Uniquely barcoded variants
    206,454

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

The figure has two parts, A and B. Part A is a schematic of the AAV2 rep and cap genes as linear DNA between left and right inverted terminal repeats. Part B depicts the library production assay. The pHelper plasmid and the pRep-Cap library are shown as circular plasmids that are transfected into HEK293 cells (shown as a flask). Cells are Iodixanol Gradient Ultracentrifuged (shown as a centrifugation tube with bands of color) and encapsidated genomes (shown as hexagonal viral particles, with 4 of the 6 containing the DNA) are separated. The Library (f sub plasmid) and Virus (f sub virus) are barcode amplified and sequenced with arrows pointing up towards these two formulas: S equals F sub virus divided by F sub plasmid and s primed equals S divided by s wild type.

Figure 1: pRep-Cap Scanning Saturation Mutagenesis (SSM) library design and use in production assay. (A) The pRep-Cap SSM library contains all possible single codon substitutions, deletions, and insertions in the AAV2 cap gene (dotted line). The total library design contains 29,400 unique amino acid variants, derived from 91,875 codon mutants, with each codon mutant represented by at least 2 unique barcodes, for a total of 206,454 variants. The 20 bp barcodes (labeled as 'BC') are located at the 3’ end of the cap gene. The rep and cap genes are flanked by ITR sequences to enable packaging of genomes into AAV capsids. (B) Transfection of the pRep-Cap SSM library into HEK293 cells along with an adenoviral helper plasmid enables production of genome-containing AAV particles.

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:

    pRep-Cap Scanning Saturation Mutagenesis (SSM) library was a gift from George Church (Addgene #199601)

  • For your References section:

    Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. Ogden PJ, Kelsic ED, Sinai S, Church GM. Science. 2019 Nov 29;366(6469):1139-1143. doi: 10.1126/science.aaw2900. PubMed 31780559
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