Journal: Cell

Article Title: Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies

doi: 10.1016/j.cell.2020.03.039

Figure Lengend Snippet: (A) Adaptable platform to integrate libraries of multiple DNA-barcoded templates into a specified genomic target and dissect human T cell behavior ex vivo or in vivo with phenotypic screens or single-cell transcriptome analysis. (B) Strategy for selective amplification and sequencing of successful on-target knock-in DNA construct barcodes. A short region of DNA mismatches is introduced into one homology arm of the HDR templates. The on-target HDR knock-ins that have both primer binding sites will be selectively amplified from genomic DNA (gDNA) or cDNA (Figures S2A). (C) Percent of amplicon sequencing reads with GFP or RFP barcodes in the indicated sorted populations 7 days after pooled knock-in of a barcoded two-member library with each construct encoding the same TCR specificity (NY-ESO-1 1G4 clone) plus a distinct additional insert (GFP or RFP) and corresponding DNA barcode. (D) Calculation of knock-in multiplicity in individual cells based on dual GFP / RFP 2-member library knock-in experiments (Figure S2C–E). The predicted percentage (right) of cells with biallelic integrations is estimated to be twice the observed percentage (left) of dual GFP+RFP+ cells as GFP+GFP+ and RFP+RFP+ biallelic cells would be observed in single positive gates. (E) Quantification of template switching in pooled knock-in experiments. The frequency of template switching at each stage of the protocol (left panel) was estimated by sorting single positive populations of T cells edited with the 2-member barcoded library (NY-ESO-1 TCR+GFP and NY-ESO-1 TCR+RFP) and using the homology arm mismatch priming strategy to selectively amplify the barcodes of on-target integrations. The predicted frequency of template switching in an arbitrarily large N-member library (N>2) can be calculated from the observed template switching for a 2-member library (STAR Methods). The pooled electroporation condition (bold) was used for subsequent pooled knock-in screens. n=2 (C–E) individual healthy human donors. See also Figure S1–S2 and Table S1

Article Snippet: The 36 constructs included in the pooled knock-in library ( Table S1 ) were designed using the Benchling DNA sequence editor, commercially synthesized as a dsDNA geneblock (IDT), and individually cloned using Gibson Assemblies into a pUC19 plasmid containing the NY-ESO-1 TCR replacement HDR sequence (except for pooled assembly conditions, whereas all geneblocks in the library were pooled prior to assembly).

Techniques: Ex Vivo, In Vivo, Amplification, Sequencing, Knock-In, Construct, Binding Assay, Electroporation