Whitehead SARS-CoV-2 Spike (S) Ectodomain and RBD Libraries
(Pooled Library #157971, #157972, #157973, #168776, #168777, #174293, #174294, #174295, #174296)

• Purpose:

  These libraries encode mutations to SARS-CoV-2 Spike ectodomain and receptor binding domain (RBD) that were screened in “Francino Urdaniz et al. One-shot identification of SARS-CoV-2 S RBD escape mutants using yeast screening”. The spike ectodomain and RBD sequences are codon optimized for S. cerevisiae.

  Spike ectodomain mutations are encoded in the S1 (Library A, B) and S2 (Library C) regions. These libraries were used to screen for mutations that stabilize the ‘up’ prefusion conformation.

  The RBD libraries include an N343Q mutation in order to make the RBD aglycosylated when expressed in S. cerevisiae. The RBD sequence has been codon-optimized for yeast expression, and encodes NNK mutations on surface residues. The depositing lab used these libraries to screen for escape mutants from a panel of monoclonal antibodies. The WT, N501Y, and E484Y libraries can be co-transformed with pJS697.

• Vector Backbone:

  Library	ID	Backbone
  SARS-CoV-2 Spike Ectodomain	157971-157973	pUC19 (Plasmid #50005)
  SARS-CoV-2 Spike RBD (WT)	168776-168777	pJS699 (Plasmid #168779)
  SARS-CoV-2 Spike RBD (N501Y)	174293-174294	pIFU001 (Plasmid #174298)
  SARS-CoV-2 Spike RBD (E484K)	174295-174296	pIFU002 (Plasmid #174299)

• Depositing Labs: Timothy Whitehead
• Publication: Francino-Urdaniz et al Cell Rep. 2021 Aug 31;36(9):109627. doi: 10.1016/j.celrep.2021.109627. Epub 2021 Aug 10.

Library Details

SARS-CoV-2 Spike Libraries

Library ID	Ectodomain Library	Spike region	Designed Mutations	Bacterial Resistance
157971	Ectodomain A	SD1	1,468	Kanamycin
157972	Ectodomain B	SD1	325	Ampicillin
157973	Ectodomain C	SD2	138	Ampicillin
Library ID	Receptor Binding Domain Library	Positions Covered	Designed Mutations	Bacterial Resistance
168776	WT RBD Library 1	333-436	1176	Kanamycin
168777	WT RBD Library 2	437-527	1323	Kanamycin
174293	E484K RBD Library 1	333-436	1120	Kanamycin
174294	E484K RBD Library 2	437-527	1260	Kanamycin
174295	N501Y RBD Library 1	333-436	1120	Kanamycin
174296	N501Y RBD Library 2	437-527	1260	Kanamycin

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.

• Volumes:
  • Ectodomain Libraries (157971-157973): ∼10 µL
  • RBD Libraries (168776-168777,174293-174296): ∼20 µL
• Concentrations:
  • Ectodomain Libraries (157971-157973): 25 ng/µL
  • RBD Libraries (168776-168777,174293-174296): 50 ng/µL

Resource Information

• Protocols:
  • Ectodomain Library Amplification and NGS Protocols (DOCX, 18 KB)
  • RBD Library Amplification and NGS Protocols (DOCX, 23 KB)
• Depositor Data:
  • List of ectodomain mutations (XLSX, 53 KB)
  • List of receptor binding domain mutations (XLSX, 17 KB)
  • Ectodomain and WT RBD Mutation Counts (XLSX, 94 KB)
  • E484K Library 1 Histogram (PNG, 180 KB)
  • E484K Library 2 Histogram (PNG, 188 KB)
  • N501Y Library 1 Histogram (PNG, 185 KB)
  • N501Y Library 2 Histogram (PNG, 181 KB)
• Reference sequences:
  • Ecto Library A Reference Sequence (FASTA, 4 KB)
  • Ecto Library B Reference Sequence (FASTA, 4 KB)
  • Ecto Library C Reference Sequence (FASTA, 4 KB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

The depositing lab recommends the following algorithm for analyzing NGS Data:: PACT (Github)

Improved mutant function prediction via PACT: Protein Analysis and Classifier Toolkit. Klesmith JR, Hackel BJ. Bioinformatics. 2019 Aug 15;35(16):2707-2712. doi: 10.1093/bioinformatics/bty1042. PubMed 30590444

bioRxiv 2021.03.15.435309.