Modular Pooled Knockin Libraries
(Pooled Libraries #207474, #207475, #207476, #207477, #207478, #207479, #207480, #207481)

• Purpose:

  Each Modular Pooled Knockin (ModPoKI) library is comprised of plasmids that include a T cell receptor (TCR) or chimeric antigen receptor (CAR) plus a gene of interest and homology arms for insertion into the TRAC locus of human T cells.

  Two sets of genes, transcription factors (TF) or cell surface receptors (SR), are available in combinations with the NY-ESO-1 TCR, CD19-BBz CAR, HA-GD2-28z CAR, or CD19-28z CAR. These libraries have been designed in a modular fashion, enabling a combinatorial set of 10,000 TF pairs (TFxTF) or screening both transcription factors and surface receptors (TF+SR) in a single screen.

  ModPoKI screening experiments with these libraries can be used to identify pathways to improve T cell immunotherapies and program other cellular functions.

• Vector Backbone:

  pUC19 - does not express Cas9

• Depositing Labs: Alexander Marson
• Publication: Blaeschke et al. Cell. 2023 Sep 14;186(19):4216-4234.e33. doi: 10.1016/j.cell.2023.08.013.

Library Details

• Species: Human

Pooled Library	Receptor	Genes Targeted	Number of Plasmids
207474	NY-ESO-1 TCR	Surface Receptors	129
207475	NY-ESO-1 TCR	Transcription Factors	100
207476	CD19-BBz CAR	Surface Receptors	129
207477	CD19-BBz CAR	Transcription Factors	100
207478	HA-GD2-28z CAR	Transcription Factors	100
207479	HA-GD2-28z CAR	Surface Receptors	129
207480	HA-GD2-28z CAR	Pairs of Transcription Factors	~10,000
207481	CD19-28z CAR	Surface Receptors and Transcription Factors	229

• Controls: Each library also includes two control plasmids encoding GFP and RFP. The TFxTF Library #207480 contains four controls: GFP-GFP, RFP-RFP, GFP-RFP and RFP-GFP.

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: ∼25 µL
• Concentration: 10 ng/µL

Resource Information

• Protocols:
  • Library Amplification Protocol (PDF, 106 KB)
  • PCR Protocol for Library Distribution Analysis (PDF, 125 KB)
  • PCR Protocol for Sequencing of Screen Samples (PDF, 131 KB)
• Depositor Data:
  • Lists of Library Barcodes (ZIP, 215 KB)
  • Library Read Counts (ZIP, 256 KB)
• Terms and Licenses:
  • UBMTA

Depositor Comments

Pooled non-viral knockin experiments can be performed by electroporating cells with the library along with Cas9 ribonucleoprotein with gRNA targeting the TRAC locus.

The plasmids consist of the respective TCR or CAR in combination with additional genes (library members), adapters, and homology arms. The surface receptor part of the libraries consists of synthetic chimeric receptors (also known as "switch receptors"), chemokine receptors, cytokine receptors, metabolic receptors, and stimulatory molecules. Barcoded adaptors facilitate pooled cloning and can also be quantified by simple amplicon sequencing.

More information is available in the publication or the preprint on bioRxiv (doi: 10.1101/2022.07.27.501186 ). Please also refer to Roth et al. 2020 Cell (doi: 10.1016/j.cell.2020.03.039 ) for more details, including bioinformatic analysis.

The NY-ESO-1 TCR sequence was provided by Antoni Ribas (University of California, Los Angeles); CD19-BBz CAR and CD19-28z CAR sequences were provided by Tobias Feuchtinger (Ludwig Maximilian University of Munich); HA-GD2-28z CAR sequence was provided by Crystal Mackall and Robbie Majzner (Stanford University).