Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Expected DNA barcodes could be selectively sequenced from cells expressing the corresponding knock-in-encoded protein products.

“We first demonstrated that expected DNA barcodes could be selectively sequenced from cells expressing the corresponding knock-in encoded protein products.”

Sorting cells with successful HDR knock-in that co-expressed GFP or RFP strongly enriched for the appropriate DNA barcode isolated and sequenced from either gDNA or cDNA.

“Sorting cells with successful HDR knock in (based on expression of the NY-ESO-1 TCR) that co-expressed either GFP+ or RFP+ strongly enriched for the appropriate DNA barcode isolated sequenced from either gDNA or cDNA (Figure 1C and Figure S2C–E).”

Primers complementary to template homology arm mismatches did not show the same enrichment for correct barcodes in sorted populations.

“However, as expected, primers complementary to the template homology arm mismatches – which should preferentially amplify non-HDR integrated templates – did not show the same enrichment for correct barcodes in the sorted populations (Figure 2E).”

Template switching varied with the stage of library production at which constructs were pooled.

“Template switching varied based on the stage of library production when constructs were pooled, and we chose to pool all subsequent knock-in construct libraries at the electroporation stage (Figure 1E).”

Relative abundances of knock-in construct barcodes could be tracked over time in an expanding population of T cells cultured in IL-2.

“Finally, we were able to readily track the relative abundances of knock-in construct barcodes over time in an expanding population of T cells cultured in interleukin 2 (IL-2).”

The platform nominated a novel TGFβR2-41BB chimeric receptor that improved solid tumor clearance.

“This platform nominated a novel TGFβR2–41BB chimeric receptor that improved solid tumor clearance.”

The homology arm mismatch sequencing strategy selectively amplified on-target barcodes using the larger 36-member pooled knock-in library.

“We next directly validated the homology arm mismatch sequencing strategy (Figure 1B) to selectively amplify on-target barcodes using the larger 36-member pooled knock-in library.”

PCR primers annealing to the inserted knock-in sequence and to the genomic flanking sequence can selectively amplify successful on-target HDR events.

“PCR primers that anneal to the inserted knock-in sequence and to the genomic flanking sequence can then selectively amplify successful on-target HDR events (Figure S2A).”

Following anti-CD3/CD28 stimulation, constructs encoding various synthetic versions of the Fas receptor were strikingly enriched.

“Following anti-CD3/CD28 stimulation, we observed a striking enrichment of constructs encoding various synthetic versions of the Fas receptor (Figure 3B).”

• AssumptionRests on an assumption: The in vitro conditions used in the pooled screens mimic aspects of tumor microenvironments.

Only about 25% of cells showed evidence of multiple integrations in the GFP and RFP duplexed barcoded library.

“Only ~25% of cells showed evidence of multiple integrations (Figure 1D and Figure S2E).”

A short DNA sequence that does not match the target genomic locus can be introduced into one homology arm without greatly reducing knock-in efficiency.

“and a short DNA sequence that does not match the target genomic locus can be introduced into one homology arm without a large reduction in knock-in efficiency (Figure 1B and Figure S2B).”

The authors created a flexible method to label and follow targeted insertions of large non-viral DNA templates in primary human T cells.

“We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates, and applied it to perform pooled knock-in screens in primary human T cells.”

• AssumptionRests on an assumption: The field lacks rapid methods to test and directly compare the functions of large DNA knock-in integrations in human primary cells.

The percentage of reads with GFP or RFP sequencing barcodes closely corresponded to the percentage of GFP- or RFP-positive cells at the protein level across four human donors.

“As expected, the percentage of reads with the GFP or RFP sequencing barcodes closely corresponded to the percentage of GFP or RFP positive cells observed at the protein level across four human donors (Figure 2D).”

A unique barcode for each insert placed within degenerate bases of the TCR-α sequence enabled rapid PCR-based DNA sequencing to measure the abundance of each on-target insert in the pooled population from genomic DNA or reverse-transcribed mRNA.

“Addition of a barcode unique for each insert within degenerate bases of the TCR-α sequence enabled a rapid DNA sequence readout by PCR of the abundance of each individual on-target insert in the pooled population by either of two complementary methods: 1) sequencing directly from genomic DNA (gDNA), or 2) sequencing from reverse transcribed mRNA (cDNA) (Figure 1B).”

For both gDNA and cDNA sequencing, when GFP+ or RFP+ cells were sorted from cells with successful on-target knock-in, the correct barcodes were selectively sequenced using primers that bound the genomic sequence at the rate predicted when accounting for multiple integrations and template switching.

“For both gDNA and cDNA sequencing conditions, when GFP+ or RFP+ cells were sorted from the cells with successful on-target knock-in (NY-ESO-1 TCR+), the correct barcodes were selectively sequenced when using primers that bound the genomic sequence at the rate predicted when taking multiple integrations and template switching into account (Figure 2E and STAR Methods).”

With pooling at the electroporation stage, only about 10% of cells showed evidence of template switching.

“With this pooling strategy only ~10% of cells showed evidence of template switching (e.g. nine in ten knock-in positive cells will have the correct combination of knock-in construct and barcode) (Figure 1E and Figure S2C–J).”

The authors developed a deep sequencing strategy that selectively captures on-target knock-in constructs while avoiding NHEJ-edited or unedited target genomic sequences, non-integrated HDR templates, or off-target integrations.

“We developed a deep sequencing strategy that selectively captures on-target knock-in constructs while avoiding NHEJ-edited or un-edited target genomic sequences, non-integrated HDR templates, or off-target integrations (Figure 1B and Figure S2A).”

All knock-in constructs in the 36-member library were well-represented across multiple pooled knock-in experiments in four independent human donors.

“Quantitative barcode sequencing demonstrated that even with the larger library all knock-in constructs were well-represented across multiple pooled knock-in experiments performed in four independent human donors (Figure 2A).”

Pooled knock-in of many unique barcoded templates into the TCR locus revealed gene constructs that improved T cell fitness in vitro and in vivo.

“Pooled knock-in of dozens of unique barcoded templates into the TCR-locus revealed gene constructs that enhanced fitness in vitro and in vivo.”

PoKI-Seq combines single-cell transcriptome analysis with pooled knock-in screening to measure cell abundance and cell state ex vivo and in vivo.

“We further developed Pooled Knock-In Sequencing (PoKI-Seq), combining single-cell transcriptome analysis and pooled knock-in screening to measure cell abundance and cell state ex vivo and in vivo.”

• AssumptionRests on an assumption: Cell abundance alone is insufficient to assess cellular function, and an ideal screening method should also assess modified cell states.

About 90% of barcodes from sorted GFP+ cells were correct.

“~ 90% of barcodes from sorted GFP+ cells were correct (Figure S2J).”

A larger library of 36 pooled templates could be introduced and tracked by their DNA barcodes.

“Quantitative barcode sequencing demonstrated that even with the larger library all knock-in constructs were well-represented across multiple pooled knock-in experiments performed in four independent human donors (Figure 2A).”

A truncated CTLA4 construct showed reduced cell killing, consistent with its impaired fitness in the pooled knock-in screens.

“In contrast, a truncated CTLA4 construct showed reduced cell killing (Figure 4D–E), consistent with its impaired fitness in the pooled knock-in screens (although diminished proliferation assessed by CFSE was not observed with this individual construct, Figure S5B).”

Sorting GFP+ or RFP+ cells from the pooled knock-in positive population strongly enriched for the expected template barcodes, confirming PoKI-Seq can accurately assign specific knock-in constructs to cells.

“Sorting GFP+ or RFP+ cells from the pooled knock-in positive population (NY-ESO-1 TCR+) strongly enriched for the expected template barcodes, confirming the ability of PoKI-Seq to accurately assign specific knock-in constructs to cells (Figure 5B).”

With exogenous TGFβ, chimeric receptors derived from TGFβR2 maintained expression of the proliferation genes MKI67 and TOP2A, whereas TGFβ suppressed these genes in cells carrying other knock-in constructs.

“For example, in the presence of exogenous TGFβ, TGFβR2-derived chimeric receptors promoted continued expression of various hallmark proliferative genes such as MKI67 and TOP2A, while these genes were inhibited by TGFβ in cells expressing other KI constructs.”

Distinct clusters of cell states emerged in PoKI-Seq of cells from two human blood donors following ex vivo stimulation with or without exogenous TGFβ, especially with TGFβ.

“Distinct clusters of cell states emerged, especially with addition of exogenous TGFβ (Figure 5D–E).”

Density plots revealed significant cell state differences in T cells modified with TGFβR2-derived constructs compared to control and non-TGFBR2-derived constructs in the stimulation plus TGFβ culture condition.

“Density plots for individual knock-in constructs revealed significant cell state differences in T cells modified with TGFβR2-derived constructs compared to control and non-TGFBR2-derived constructs in the stimulation plus TGFβ culture condition (Figure 5G).”

The TGFβR2-41BB construct increased target in vitro cancer cell killing in T cells from four human blood donors across a range of effector to target cell ratios.

“Although this was not the phenotype initially tested in the pooled screens, the TGFβR2–41BB increased target in vitro cancer cell killing in T cells from four human blood donors across a range of effector to target cell ratios (Figure 4D–E).”

A truncated CTLA4 protein decreased T cell fitness in the same conditions across four donors.

“In contrast, a truncated CTLA4 protein decreased T cell fitness in the same conditions across four donors”

The pooled knock-in screens confirmed the functional effects of a recently described truncated Fas receptor included as a positive control.

“The pooled knock-in screens confirmed the functional effects of a recently described truncated Fas receptor (Yamamoto et al., 2019) included as a positive control”

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The screens identified novel synthetic Fas switch receptors, including one with a 4-1BB intracellular domain, that promoted T cell fitness in stimulated conditions.

“also identified novel synthetic Fas switch receptors, including one with a 4–1BB intracellular domain, that also promoted T cell fitness in stimulated conditions (Figure 3B).”

In the presence of exogenous TGFβ, the TGFβR2-41BB construct rescued impaired cancer cell killing across experiments from four healthy human donors.

“In the presence of exogenous TGFβ, the TGFβR2–41BB construct successfully rescued the impaired cancer cell killing across experiments performed from four healthy human donors (Figure 4F).”

The overall proportion of cells assigned two knock-in constructs closely corresponded with predicted frequencies based on GFP/RFP 2-member library experiments.

“The overall proportion of cells assigned two knock-in constructs (Figure 5C) closely corresponded with predicted frequencies based on GFP/RFP 2-member library experiments (Figure 1D).”

Addition of TGFβ1 gave cells expressing engineered TGFβR receptors a selective fitness advantage, including a previously described truncated TGFβR2 receptor and a novel chimeric TGFβR2-41BB receptor.

“Finally, addition of the immunosuppressive cytokine TGFβ1 gave cells expressing engineered TGFβR receptors a selective fitness advantage, including both a previously described truncated TGFβR2 receptor (Gorelik and Flavell, 2000; Kim et al., 2017; Kloss et al., 2018) included as a positive control and a novel chimeric TGFβR2–41BB receptor (Figure 3D and Figure S4D–E).”

• cites Gorelik and Flavell, 2000Not checked yet

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• cites Kim et al., 2017 sourceNot checked yet

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• cites Kloss et al., 2018 sourceNot checked yet

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Cells possessing TGFβR2-derived constructs were more likely to be found in clusters otherwise associated with the stimulation-only condition rather than the stimulation + TGFβ condition, including cluster 8 (proliferation) and cluster 12 (cell killing).

“Specifically, cells possessing TGFβR2-derived constructs were more likely to be found in clusters otherwise associated with the stimulation-only condition rather than the stimulation + TGFβ condition, including cluster 8, characterized by genes associated with cell proliferation, and cluster 12, characterized by genes associated with cell killing (Figures 5E, H and S6E–F).”

In vivo screens had greater variance between replicates and donors than in vitro screens.

“Due to the lower number of human T cells isolatable from individual solid tumor xenografts (generally 10,000–20,000 cells per mouse), greater variance between replicates and donors was observed in the in vivo screens than in the in vitro screens (Figure S7B–C).”

Single-cell template barcode assignments were confirmed further as transcripts encoded by corresponding knock-in constructs tended to be expressed at increased levels.

“Single cell template barcode assignments were confirmed further as transcripts encoded by corresponding knock-in constructs tended to be expressed at increased levels, similar to what had been observed at the protein level with delivery of individual knock-in constructs (Figure 4B and Figure S6D).”

The TCF7 cell population did not improve tumor control relative to tNGFR controls.

“Consistent with TCF7 promoting TIL abundance, but not promoting mature effector cytokine expressing cells, the TCF7 cell population did not enhance tumor control relative to tNGFR controls (Figure 7B).”

When sorted GFP or RFP positive cells were assigned two knock-in constructs, at least one of the assigned constructs was almost always either GFP or RFP respectively.

“As expected, when sorted GFP or RFP positive cells were assigned two knock-in constructs, at least one of the knock-in constructs assigned was almost always either GFP or RFP respectively (Figure 5C).”

Each knock-in construct caused distinct enrichment patterns in individual cell clusters in both TCR stimulation and stimulation plus TGFβ conditions that broadly corresponded to results from the in vitro pooled knock-in screens.

“Each knock-in construct caused distinct enrichment patterns in individual cell clusters in both TCR stimulation and stimulation plus TGFβ conditions that broadly corresponded to results from the in vitro pooled knock-in screens (Figure S6E).”

TGFβR2-derived knock-in constructs were relatively depleted from cells in clusters otherwise promoted by TGFβ treatment (Clusters 2, 4 and 6).

“Similarly, the TGFβR2-derived knock-in constructs were relatively depleted from cells in the clusters otherwise promoted by TGFβ treatment (Clusters 2, 4 and 6) (Figure 5H).”

The TGFβR2-41BB construct markedly reduced tumor sizes compared with TCR+tNGFR (P < 0.0001 at day 33).

“In contrast, the TGFβR2–41BB receptor construct that promoted abundance and effector cytokine gene expression in TILs markedly reduced tumor sizes (P < 0.0001 at day 33 vs TCR+tNGFR).”

The TCF7 knock-in construct, which had only mild and non-significant effects in vitro, increased T cell accumulation in the tumor microenvironment across four donors.

“The TCF7 knock-in construct that only showed mild effects in vitro (not reaching statistical significance) promoted accumulation of T cells in the tumor microenviroment across four donors (Figure 6B).”

TCR stimulation without anti-CD28 co-stimulation selected for various chimeric receptors with intracellular CD28 domains such as TIM3-CD28 and CTLA4-CD28.

“TCR stimulation without anti-CD28 co-stimulation selected for various chimeric receptors with intracellular CD28 domains such as TIM3-CD28 and CTLA4-CD28 (Figure S4C).”

Exposure to TGFβ caused the largest transcriptional changes among the conditions tested.

“Hierarchical clustering revealed that exposure to TGFβ drove the greatest transcriptional changes (Figure 5I and Figure S6G).”

The lead construct showed markedly enhanced clearance of an in vivo solid tumor model.

“This lead construct indeed showed markedly enhanced clearance of an in vivo solid tumor model.”

Increased cell expansion induced by the TGFβR2-41BB construct correlated with evidence of increased cell proliferation in the presence of TGFβ, measured by CFSE dilution relative to control cells.

“Increased cell expansion induced by the TGFβR2–41BB construct correlated with evidence of increased cell proliferation in the presence of TGFβ (measured by CFSE dilution relative to control cells) (Figure 4C and Figure S5B).”

The measured template-switching rate for the pooled electroporation condition used in the study was about 10%.

“the rate of template switching in the pooled electroporation condition used throughout the study was determined to be ~10% (Figure 1E)”

The estimated rate of biallelic knock-in was about 25% among cells positive for knock-in.

“Biallelic knock-in rate was estimated to be ~25% of total knock-in positive cells (Figure 1D and Figure S2E)”

Engineered cell phenotypes could be quantitatively tracked in pooled knock-in experiments through DNA barcode sequencing.

“These results showed that engineered cell phenotypes could be quantitatively tracked in pooled knock-in experiments through DNA barcode sequencing.”

Pooled screening approaches have been challenged by template switching that complicates interpretation of DNA barcode readouts.

“Pooled screening approaches have been challenged by template switching that complicate interpretation of DNA barcode readouts (Hanna and Doench, 2018; Hegde et al., 2018; Sack et al., 2016).”

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• cites Hegde et al., 2018 sourceNot checked yet

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• cites Sack et al., 2016 sourceNot checked yet

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Most homology arm sequences of an HDR template are not incorporated into on-target HDR integrations.

“Briefly, the majority of a HDR template’s homology arm sequences are not incorporated with on-target HDR integrations (Paquet et al., 2016)”

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Targeted integration of antigen receptor genes into defined genomic loci has potential advantages.

“More recent work has revealed potential advantages of targeted integrations of antigen receptor genes into defined genomic loci (Eyquem et al., 2017), which can be achieved with programmable nucleases such as CRISPR-Cas9 along with viral or non-viral knock-in templates (Gwiazda et al., 2016; Eyquem et al., 2017; Roth et al., 2018).”

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Multiple barcoded templates introduced into the same cell could complicate interpretation of each one's effects.

“In addition, multiple barcoded templates introduced in the same cell could complicate interpretation of each one’s effects (Feldman et al., 2018; Roth et al., 2018).”

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Excessive antigen density and kinetics of antigen exposure in solid tumors can drive T cell exhaustion or dysfunction.

“The excessive antigen density and kinetics of antigen exposure in solid tumors can drive exhaustion or dysfunction of T cells (Khan et al., 2019; Wherry, 2011).”

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Suppressive cytokines such as TGFβ can be co-opted by tumors to inhibit T cell function.

“Suppressive cytokines such as TGFβ can be co-opted by tumors to inhibit T cell function (Dahmani and Delisle, 2018; Thomas and Massagué, 2005).”

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Tregs, MDSCs, and dendritic and stromal cell populations within the tumor can deprive T cells of necessary co-stimulatory signals or induce inhibitory signals.

“Tregs, myeloid derived suppressor cells (MDSCs), and dendritic and stromal cell populations within the tumor can deprive T cells of necessary co-stimulatory signals or induce inhibitory signals (Chen and Mellman, 2013; Ostrand-Rosenberg et al., 2012).”

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Next-generation cellular immunotherapies will likely need enhanced function to overcome solid tumor challenges.

“Next-generation cellular immunotherapies will likely need to be engineered with enhanced function to overcome these challenges presented by solid tumors (Rafiq et al., 2019).”

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• AssumptionRests on an assumption: CAR T cells have not proved effective for solid tumors.

The translational potential for knock-in of large gene cassettes to rewrite cell programs is greater than that of gene ablation.

“While gene ablation can enhance cell therapies, the translational potential for knock-in of large gene cassettes to re-write cell programs is even greater.”

Coupling pooled CRISPR gene knockouts with single-cell RNA sequencing provides a powerful approach to assess the effect of each gene perturbation on cell state.

“Coupling pooled CRISPR gene knockouts with single cell RNA sequencing provides a powerful approach to assess the effect of each gene perturbation on cell state (Adamson et al., 2016; Datlinger et al., 2017; Dixit et al., 2016; Jaitin et al., 2016).”

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• cites Datlinger et al., 2017 sourceNot checked yet

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• cites Dixit et al., 2016 sourceNot checked yet

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Pooled knock-in screening allows parallel rewriting of endogenous genetic sequences to speed discovery of knock-in programs for cell therapies.

“Pooled knock-in screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knock-in programs for cell therapies.”

Barcoded pools of non-viral HDR templates can now be used to explore in vitro and in vivo functions of diverse sequence variants at targeted coding and non-coding elements in primary human cell genomes.

“Barcoded pools of non-viral HDR templates can now be used to explore the in vitro and in vivo functions of diverse sequence variants at targeted coding and non-coding elements in the genomes of primary human cells.”

Pooled knock-in screening combined with scRNA-seq can rapidly identify new DNA sequences with potential to improve therapeutic T cell functions.

“Taken together, pooled knock-in screening combined with scRNA-seq can rapidly reveal new DNA sequences with potential to enhance therapeutic T cell functions.”

Effective barcoding of targeted knock-in sequences allowed single-cell transcriptome readouts in a pool of modified cells to assess each construct's effect on cell state, a method termed PoKI-seq.

“Effective barcoding of targeted knock-in sequences also allowed single-cell transcriptome readouts in a pool of modified cells to assess the effects of each knock-in construct on cell state, a method we term Pooled Knock-In Sequencing (PoKI-seq).”

Pooled knock-in screens highlight condition-specific functional enhancements conferred by synthetic proteins or forced expression of key regulators.

“These data from pooled knock-in screens highlight condition-specific functional enhancements conferred by synthetic proteins or forced expression of key regulators.”

Targeted non-viral pooled knock-in screens have notable advantages over other systems that could be used to discover effects of gene gain-of-function.

“Targeted non-viral pooled knock-in screens have notable advantages over other systems that potentially could be used to discover effects of gene gain-of-function.”

• AssumptionRests on an assumption: Technical challenges have limited CRISPRa applications in therapeutically relevant primary human cell types.
• AssumptionRests on an assumption: CRISPRa can only induce natural gene products encoded in the genome and does so with variable efficiency.
• AssumptionRests on an assumption: A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.
• AssumptionRests on an assumption: Most over-expression studies have been performed with lentiviral or retroviral vectors.

The pooled screens, although focused on cell-intrinsic effects on T cell fitness, also identified novel gene constructs that can enhance in vitro anti-cancer cell efficacy.

“Although the pooled screens focused on cell-intrinsic effects on T cell fitness, they also successfully identified novel gene constructs that can enhance in vitro anti-cancer cell efficacy.”

The paper overcame technical limitations in tracking targeted genomic integrations with a widely adaptable technology to barcode and track on-target integrations of HDR templates in a pooled library.

“Here we overcame these technical limitations with a widely adaptable technology to barcode and track on-target integrations of HDR templates in a pooled library.”

• AssumptionRests on an assumption: A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.

The results of the pooled targeted knock-in screens were reproducible across multiple biological replicates, technical replicates, sorting conditions and pooling conditions.

“The results of these pooled targeted knock-in screens were reproducible across multiple biological replicates, technical replicates, sorting conditions and pooling conditions (Figure S4F–H).”

Each knock-in construct can be tracked in the targeted cell population under diverse in vitro and in vivo conditions to reveal the most effective modifications.

“Each knock-in construct can be tracked in the targeted cell population under diverse in vitro and in vivo conditions to reveal the most effective modifications.”

TCF7 expression is lost when cells differentiate into effector cells.

“but TCF7 expression was lost upon differentiation into effector cells (Siddiqui et al., 2019).”

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PoKI-Seq can reveal target gene pathways and context-dependent cell-state changes caused by individual knock-in constructs.

“More generally PoKI-Seq was able to reveal target gene pathways and context-dependent changes in cell state modulated by individual knock-in constructs.”

The pooled knock-in platform offers a new opportunity to systematically screen large gene constructs that can be added to T cells to modulate critical functions for effective cell therapies.

“The pooled knock-in platform presented here offers a new opportunity to screen systematically for large gene constructs that can be added to a T cell to modulate critical functions for effective cell therapies.”

• AssumptionRests on an assumption: The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.

PoKI-Seq can phenotype individual cells with either single or multiple knock-in constructs.

“Overall, these experiments confirmed the molecular biology of PoKI-seq and also demonstrated its ability to phenotype individual cells with either single or multiple knock-in constructs.”

Non-viral pooled knock-in screening will accelerate discovery and development of synthetic DNA sequences to reprogram specificity and function of adoptive cellular therapies.

“Non-viral pooled knock-in screening will accelerate the discovery and development of synthetic DNA sequences to reprogram the specificity and function of adoptive cellular therapies.”

• AssumptionRests on an assumption: The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.

The pooled library-based approach can identify individual knock-in sequences to engineer T cell function.

“These findings validated the pooled library-based approach to identify individual knock-in sequences to engineer T cell function.”

PoKI-seq offers a complementary single-cell approach to study large knock-in perturbations in pooled experiments.

“PoKI-seq offers a complementary single-cell approach to study large knock-in perturbations in pooled experiments.”

Knock-in of large genetic constructs enables engineering synthetic receptors that re-wire the signal T cells receive upon TGFβ exposure in the tumor microenvironment, including switching suppressive TGFβ signaling to stimulatory 41BB signaling.

“Knock-in of large genetic constructs enables the engineering of synthetic receptors to re-wire the signal T cells receive when exposed to TGFβ in the tumor microenvironment, including switching the suppressive TGFβ signal to a stimulatory 41BB signal.”

PoKI-Seq confirmed that multiple TGFβR2-derived receptors share biological effects, converting a suppressive signal into one that promotes proliferation and an effector cell state.

“PoKI-Seq confirmed the shared biological effects of multiple TGFβR2-derived receptors, which convert a suppressive signal into a signal that promotes cell proliferation and an effector cell state.”

These sources of variability could complicate interpretation of pooled screens by introducing false positives and false negatives.

“These sources of variability could complicate interpretation of pooled screens, introducing both false positives and false negatives.”

The decreased fitness from truncated CTLA4 was likely due to competition for CD28 co-stimulatory signaling.

“likely due to competition for CD28 co-stimulatory signaling (Figure 3B).”

The preclinical success of a novel construct nominated by pooled knock-in screens highlights the power of the platform.

“The preclinical success of a novel construct nominated by pooled knock-in screens highlights the power of the platform.”

Complementary in vitro and in vivo assays are powerful for assessing synthetic gene functions.

“These results underscore the power of complementary in vitro and in vivo assays to assess synthetic gene functions.”

The metrics established the fidelity of barcode assignment to single cell transcriptomes for the PoKI-Seq platform.

“These metrics together established the fidelity of barcode assignment to single cell transcriptomes for the PoKI-Seq platform.”

For a two-member library, the actual switching rate Y is given by Y = 2/(1/X - 1), where X is the observed rate.

“Simplifying the equation yields: 3)Y/X=2+2*X+2*X2+2*X3+…+2*Xn 4)Y/X=2+Y 5)(Y/X)−Y=2 6)Y*(1/X−1)=2 7)Y=2/(1/X−1)”

• AssumptionThe authors state this as an assumption: Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.
• AssumptionThe authors state this as an assumption: A template that has switched once is equally likely to switch again.

Prior to template switching, 1000 of 1250 alleles are expected to contain the correct construct or barcode.

“Therefore, out of 1250 total alleles, 1000 would be predicted to contain the correct knock-in construct/barcode before accounting for template switching.”

• AssumptionThe authors state this as an assumption: The knock-in library is arbitrarily large.

In an arbitrarily large library, switching to templates with the same barcode becomes negligible, so observed and actual template switching rates converge.

“In a library of arbitrarily large size, there will be no significant switching with templates containing the same barcode, and thus the observed and actual amounts of template switching will converge.”

Overall, 900 of 1250 alleles, or 72%, are predicted to contain the correct knock-in barcode.

“In total then, 900 out of 1250 alleles, or 72% of alleles, will contain the correct knock-in barcode.”

In biallelic knock-in cells, half of the alleles are expected to carry a different knock-in construct or barcode than the sorted construct, assuming an arbitrarily large library.

“Half of the alleles in biallelic knock-in cells will have a different knock-in construct/barcode than the sorted (GFP) knock-in construct/barcode, assuming an arbitrarily large knock-in library.”

• AssumptionThe authors state this as an assumption: The knock-in library is arbitrarily large.

At a 10% switching rate, 100 of the 1000 correct-construct alleles are expected to have a different barcode.

“Assuming a 10% rate of template switching, 100 out of 1000 alleles with the correct knock-in construct (GFP) will contain a barcode for a different construct.”