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

• 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)

• Depositing Labs: George Church
• Publication: Ogden et al Science. 2019 Nov 29;366(6469):1139-1143. doi: 10.1126/science.aaw2900.

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

• Protocols:
  • pRep-Cap SSM Library Amplification and Sequencing Protocol (DOC, 36 KB)
• Depositor Data:
  • pRep-Cap SSM barcode counts density plot (PNG, 117 KB)
  • pRep-Cap SSM barcode to mutations (CSV, 8.4 MB)
• Terms and Licenses:
  • UBMTA

Depositor Comments

Deconvolution algorithms for analyzing NGS data is available on GitHub .

• 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.