Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

May combine two claims.

In cells expressing Pro-Code 3 linked to a CD4 gRNA, 85% were CD4 negative, whereas cells with unrelated gRNAs were almost all CD4 positive.

“in cells expressing Pro-Code 3, which was linked to a gRNA targeting the CD4 gene, 85% of the cells were CD4 negative, whereas cells expressing Pro-Codes linked to gRNAs targeting unrelated genes were almost all CD4 positive”

May combine two claims.

When treated with GM-CSF, 60-70% of cells in clusters encoding IL6R, IFNGR1, and IFNGR2 gRNAs upregulated pSTAT5, but only 30-40% of cells in CD116 gRNA clusters did.

“When the cells were treated with GM-CSF, 60–70% of the cells in the clusters encoding gRNAs targeting IL6R, IFNGR1, and IFNGR2 upregulated pSTAT5, but only 30–40% of the cells in the Pro-Code clusters encoding CD116 gRNAs upregulated pSTAT5”

May combine two claims.

All 10 epitope tags were detected with clear signal over background, and all epitope-positive cells were positive for NGFR.

“All 10 epitope tags were detected with a clear signal over background, and all of the epitope-positive cells were positive for NGFR”

May combine two claims.

The same initial mix of 120 Pro-Code subpopulations developed into heterogenic tumors, with 10 populations accounting for up to 50% of total cells.

“The same initial mix of 120 Pro-Code subpopulations developed into heterogenic tumors, in which 10 populations accounted for up to 50% of the total cell number.”

May combine two claims.

Fourteen 4-epitope Pro-Codes were generated, all 10 epitopes were detected, cells were positive for 4 epitopes, and all 14 populations were identified.

“We detected all 10 epitopes, and cells were positive for 4 epitopes. This enabled us to identify all 14 4-epitope Pro-Code populations”

May combine two claims.

HLA-A was expressed by >90% of cells in each Pro-Code/CRISPR cluster except those with Pro-Codes 23 and 24 linked to B2m gRNAs, where 45% and 80% were HLA-A negative.

“HLA-A (MHC class I) was expressed by >90% of cells in each Pro-Code/CRISPR cluster, except those expressing Pro-Codes 23 and 24, which were linked to gRNAs targeting B2m; in these populations 45% and 80% of THP1 were HLA-A negative, respectively.”

May combine two claims. The quote only partly states this claim.

When the 96 Pro-Code/CRISPR library was produced as a pool with a low-homology GFP spike, all 96 Pro-Code populations were resolved and specific protein loss was consistently observed on a high percent of cells expressing the cognate gRNA.

“Similar to the library made with individually packaged vectors, we resolved all 96 Pro-Code populations, and consistently observed loss of a specific protein on a high percent of cells expressing a Pro-Code linked to a gRNA targeting the cognate gene”

May combine two claims.

Single-cell analysis indicated significant heterogeneity between cells even within the same Pro-Code cluster, despite bulk analysis showing major reduction in pSTAT signaling downstream of the targeted receptor.

“Thus, while the bulk analysis indicated a major reduction in pSTAT signaling downstream of the receptor targeted by a specific CRISPR, single cell analysis indicated there was significant heterogeneity between cells even within the same Pro-Code cluster.”

Probably not a substantial claim: may not pass S3 (load-bearing).

There was little evidence of knockout for some gRNAs, consistent with known variability in CRISPR efficiency between gRNAs.

“There was little evidence of KO for some gRNAs, consistent with the known variability in CRISPR efficiency between gRNAs.”

• AssumptionRests on an assumption: CRISPR editing efficiency varies between gRNAs.

The quote only partly states this claim.

All 96 Pro-Code populations were resolved and clustered after transduction of THP1-Cas9 with 96 Pro-Code/CRISPR vectors.

“All 96 Pro-Code populations were resolved and clustered.”

The quote only partly states this claim.

Unsupervised clustering resolved 120 distinct Pro-Code populations across 293T, THP1, Jurkat, and 4T1 cells, each corresponding to one Pro-Code.

“Unsupervised clustering resolved 120 distinct populations (Figure 1F-I), with each population corresponding to one Pro-Code”

May combine two claims.

Using 14 antibodies, the authors detected 364 Pro-Code populations, establishing the largest set of protein-based reporters.

“Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters.”

May combine two claims.

The frequency of each Pro-Code population ranged from 0.1% to 3%, with 65% between 0.4-1.5%, close to the expected 0.83% if equimolar.

“The frequency of each population ranged from 0.1% to 3%, with the majority of Pro-Code populations (65%) being between 0.4–1.5% (Figure S2D), which is close to the expected frequency of 0.83% if each of the 120 Pro-Codes was in equimolar concentration.”

May combine two claims.

When treated with IFNγ, 70% of cells in Pro-Code clusters linked to CD116 and IL6R gRNAs had increased pSTAT1, whereas only ~25% of cells in IFNGR1/IFNGR2 clusters did.

“When we treated cells with IFNγ, 70% of the cells in the Pro-Code clusters linked to gRNAs targeting CD116 and IL6R had increased pSTAT1, whereas in the Pro-Code clusters linked to gRNAs targeting IFNGR1 and IFNGR2, only ~25% of the cells had increased pSTAT1”

May combine two claims.

Some Pro-Code populations were abundant in every tumor but varied in proportion, while others were abundant only in a single tumor.

“Some Pro-Code populations were abundant in every tumor (e.g. Pro-Codes 108 and 21), but their proportion within each tumor varied greatly, whereas other Pro-Code populations were only abundant in a single tumor, such as Pro-Code 6”

May combine two claims.

Using 14 epitopes, 364 3-epitope Pro-Code vectors were generated and all 364 expressing populations were identified and clustered in 293T cells.

“Using an expanded set of 14 epitopes, we generated 364 3-epitope Pro-Code vectors, and introduced them in to 293T. The cells were stained for NGFR and all 14 epitopes, analyzed by CyTOF, and all 364 Pro-Code expressing populations were readily identified and clustered”

The quote only partly states this claim.

In cultures containing activated Jedi T cells, populations of 4T1-GFP emerged that had downregulated H-2Kd or GFP and escaped T cell killing, even with individual scrambled gRNA vectors.

“As we observed with the pool of vectors, in cultures containing activated Jedi T cells there emerged populations of 4T1-GFP that had downregulated H-2Kd or GFP and escaped T cell killing”

May combine two claims.

Cells exposed to activated Jedi T cells upregulated both MHC-I and PD-L1.

“As expected, cells exposed to activated Jedi T cells upregulated both MHC-I and PD-L1”

May combine two claims.

Adding a low-homology lentiviral plasmid during vector production reduces barcode swapping, since knockout efficiency was similar for libraries made with individually packaged vectors and those made as a pool with the low-homology vector.

“We also found that introducing a plasmid encoding an LV with low-homology to the library during vector production reduces barcode swapping, as the efficiency of KO was similar between Pro-Code/CRISPR libraries made with individually packaged vectors or vectors produced as a pool with the low-homology vector.”

May combine two claims.

Whereas <0.1% of control 4T1-GFP cells remained in Jedi co-cultures, ~4% of Rtp4 CRISPR and 10% of Psmb8 CRISPR 4T1-GFP cells remained.

“Whereas <0.1% of control 4T1-GFP cells remained in the Jedi co-cultures, ~4% of the Rtp4 CRISPR and 10% of the Psmb8 CRISPR 4T1-GFP cells remained.”

May combine two claims.

Pro-Codes directly measure the frequency of each CRISPR-carrying cell in a population, whereas DNA barcodes infer vector percentage from sequence frequency.

“as opposed to DNA barcodes in which the percent of each vector is presumed from sequence frequency, with the Pro-Codes, the frequency of each CRISPR-carrying cell within a population is directly determined.”

May combine two claims.

Co-culture of 4T1 cells with activated CD8+ Jedi T cells caused selective killing of GFP+ cells, quantifiable by flow cytometry.

“When the cells were co-cultured with activated CD8+ Jedi T cells there was selective killing of the GFP+ cells, which could be quantified by flow cytometry”

May combine two claims.

Cells carrying Ifngr2 CRISPR did not upregulate MHC-I in response to IFNγ but still expressed high levels of MHC-I comparable to activated Jedi T cells.

“Though the cells carrying the Ifngr2 CRISPR did not upregulate MHC-I in response to IFNγ, the cells still expressed high levels of MHC-I (Figure S6B). Indeed, the levels of MHC-I were comparable to the activated Jedi T cells.”

May combine two claims.

The frequency of 4T1-Cas9-mCherry cells expressing Psmb8 and Rtp4 gRNAs did not significantly change, indicating enrichment was dependent on antigen-specific T cell killing.

“The frequency of 4T1-Cas9-mCherry cells expressing Psmb8 and Rtp4 gRNAs did not significantly change, indicating enrichment was dependent on antigen-specific T cell killing.”

May combine two claims.

A common feature of these residual cells was decreased GFP and/or MHC-I.

“Interestingly, a common feature of these cells was decreased gFp and/or MHC-I”

May combine two claims.

Sanger sequencing and qPCR confirmed the Rtp4 gene had been mutated and was no longer expressed.

“we used Sanger sequencing and qPCR and confirmed the Rtp4 gene had been mutated and was no longer expressed”

May combine two claims.

Pro-Codes enable analysis of millions of single cells with accurate protein-level quantification.

“The Pro-Codes makes it possible to analyze millions of single cells with precise quantification of protein levels.”

May combine two claims.

In cells encoding Ifngr2 CRISPRs, MHC-I levels were normal at steady state, but MHC-I did not increase in Jedi co-cultures as it did in cells carrying unrelated CRISPRs.

“In cells encoding Ifngr2 CRISPRs, there were normal levels of MHC-I expression in steady-state, but the expression of MHC-I on these cells did not increase in the Jedi co-cultures, as it did in cells carrying unrelated CRISPRs.”

May combine two claims.

Many resistant cells had reduced MHC-I or reduced target antigen GFP.

“many resistant cells had lower levels of MHC-I or the target antigen, GFP.”

May combine two claims.

The results support a model in which clonal growth was largely stochastic and not impacted by the Pro-Codes.

“These results support a model in which clonal growth was largely stochastic and not impacted by the Pro-Codes”

Classified as probably not a substantial claim.

Previous screens used antigen-specific T cells targeting model tumor-associated antigens such as OVA, gp100, and NY-ESO-1.

“In previous screens, antigen-specific T cells targeting model tumor associated antigen (TAA), such as OVA, gp100, and NY-ESO-1, were used (Manguso et al., 2017; Pan et al., 2018; Patel et al., 2017).”

• cites Manguso et al., 2017 sourceNot checked yet

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• cites Pan et al., 2018 sourceNot checked yet

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• cites Patel et al., 2017 sourceNot checked yet

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May combine two claims.

Single-cell RNA sequencing CRISPR screens have restrained cell throughput and cannot measure important protein-level phenotypic information such as signaling alterations.

“However, the cell throughput is relatively restrained, and important protein-level phenotypic information, such as signaling alterations, cannot be measured.”

May combine two claims.

DNA barcoding has major limitations, including a bulk-cell readout that prevents ready single-cell analysis.

“DNA barcoding has major limitations. One of the most significant is that the read-out is performed on bulk cells, which means single cells cannot be readily analyzed.”

May combine two claims.

As few as 10 epitopes arranged in sets of 3 create 120 unique combinations, and 20 epitopes in 7 positions create 77,520 combinations.

“Using this strategy, as few as 10 epitopes can be arranged in sets of 3 to create 120 unique combinations, and with just 20 epitopes and 7 positions, 77,520 combinations can be generated.”

Classified as probably not a substantial claim.

Barcode swapping can be reduced by co-packaging libraries with a low homology transfer vector.

“Recently, it was reported in pre-print studies that swapping can be reduced by co-packaging libraries with a low homology transfer vector (Adamson et al., 2018; Feldman et al., 2018).”

• cites Adamson et al., 2018Not checked yet

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• cites Feldman et al., 2018Not checked yet

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May combine two claims.

DNA barcoding does not enable direct cell phenotyping; instead, phenotype is inferred from changes in barcode frequency.

“Another limitation is that DNA barcoding does not enable cells to be directly phenotyped. Instead, the phenotype associated with each gene perturbation is inferred from changes in barcode frequency.”

Classified as probably not a substantial claim.

Linear epitopes are needed to assemble the barcodes because they can be encoded by short sequences.

“We reasoned linear epitopes would be needed to assemble the barcodes because they can be encoded by a short sequence (18–42 nucleotides).”

Classified as probably not a substantial claim.

Packaging vector pools together can lead to varying degrees of barcode swapping.

“As it has recently been reported that packaging vector pools together can lead to varying degrees of barcode swapping (Hill et al., 2018; Sack et al., 2016)”

• cites Hill et al., 2018 sourceNot checked yet

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• cites Sack et al., 2016 sourceNot checked yet

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Probably not a substantial claim: may not pass S4 (based).

Cancer cells can alter gene expression to resist being killed.

“However, cancer cells can alter gene expression to resist being killed.”

May combine two claims.

This heterogeneity reflects biological differences between cells in response to cytokine stimulation and cell-to-cell heterogeneity in CRISPR knockout.

“This heterogeneity reflects biological differences between cells in their response to cytokine stimulation, but also reveals cell-to-cell heterogeneity in CRISPR KO, as observed in our studies above measuring the protein levels of the gene targeted by specific CRISPRs.”

May combine two claims.

Pro-Code/CRISPR libraries can be produced as a pool and function at high efficiency.

“These results indicate Pro-Code/CRISPR libraries can be produced as a pool and function at high efficiency”

May combine two claims.

Loss of HLA in B2m-targeted clusters represents a downstream phenotype of B2m knockout because B2m is required for HLA stability.

“As B2m is required for HLA stability, the loss of HLA in these clusters represents a downstream phenotype of B2m KO.”

• AssumptionRests on an assumption: B2m is required for HLA stability.

May combine two claims.

Pro-Codes are not differentially rejected and can be used in vivo.

“This demonstrates the Pro-Codes are not differentially rejected, and can be used in vivo.”

May combine two claims.

Pro-Codes enable direct single-cell phenotypic analysis of signaling pathways in CRISPR screens, which is not feasible with DNA or RNA level analysis.

“Together, these results demonstrate Pro-Codes enable direct single cell phenotypic analysis of signaling pathways in CRISPR screens, which is not feasible with DNA or RNA level analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The IFNγ pathway has been linked to clinical response to checkpoint inhibitors.

“The IFNγ pathway has been implicated as a key component in the clinical response to checkpoint inhibitors (Minn and Wherry, 2016).”

• cites Minn and Wherry, 2016Not checked yet

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May combine two claims.

While far fewer barcodes than DNA, Pro-Codes provide an order of magnitude more barcodes than current protein reporters.

“While this is far fewer barcodes than achieved with DNa, it is an order of magnitude greater than what currently exists with protein reporters.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Multiple groups have used scRNA-seq in pooled screens to gain more comprehensive phenotyping and single-cell resolution.

“Several groups have incorporated scRNA-seq into pooled screens to obtain more comprehensive phenotyping than previously possible, and to achieve single cell resolution (Adamson et al., 2016; Datlinger et al., 2017; Dixit et al., 2016; Jaitin et al., 2016).”

• cites Adamson et al., 2016 sourceNot checked yet

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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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Classified as probably not a substantial claim. May combine two claims.

Swapping arises when two different vector genomes are packaged in the same virion and template switching occurs during reverse transcription.

“Swapping occurs when two different vector genomes are packaged in the same virion, and there is template switching during reverse transcription (King et al., 2008).”

• cites King et al., 2008 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Mutations in IFNGR1 and JAK, part of IFNγ signaling, have been found in patients who developed resistance to checkpoint inhibitors.

“Mutations in IFNGR1 and JAK, a component of the IFNγ signaling pathway, have been found in patients presenting resistance to checkpoint inhibitors (Gao et al., 2016; Zaretsky et al., 2016).”

• cites Gao et al., 2016 sourceNot checked yet

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• cites Zaretsky et al., 2016 sourceNot checked yet

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May combine two claims.

The RFP+ cells serve as an internal control of non-TAA-expressing cells and enable distinction between effects of a specific knockout on cell fitness versus T cell sensitivity.

“The RFP+ cells serve as an internal control of non-TAA expressing cells, and enables distinction between the effects of a specific KO on cell fitness versus T cell sensitivity.”

May combine two claims.

By marking hundreds of CRISPR-expressing populations and measuring many protein markers, Pro-Codes expand the types of pooled genetic screens and help facilitate gene function annotation.

“By being able to mark 100s of different CRISPR-expressing populations and measure many protein markers, Pro-Codes expand the types of pooled genetic screens that can be performed, and will help facilitate the annotation of gene functions.”

Classified as probably not a substantial claim. May combine two claims.

Psmb8 (Lmp7) is part of the immunoproteasome, which generates peptides for MHC-I.

“Psmb8 (also known as Lmp7) is a component of the immunoproteasome, which functions in generating peptides for MHC-I (Basler et al., 2013)”

• cites Basler et al., 2013 sourceNot checked yet

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May combine two claims.

These findings are consistent with clinical data correlating resistance to checkpoint inhibitors with mutations in B2m and IFNγ pathways and with recent genome-wide CRISPR screening data.

“These findings are consistent with emerging clinical data correlating resistance to checkpoint inhibitors with mutations in the B2m and IFNγ pathways (Gao et al., 2016; Zaretsky et al., 2016), and with recent genome-wide CRISPR screening data (Patel et al., 2017).”

• cites Gao et al., 2016 sourceNot checked yet

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• cites Zaretsky et al., 2016 sourceNot checked yet

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• cites Patel et al., 2017 sourceNot checked yet

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May combine two claims.

Barcode swapping can happen in pooled retroviral vector libraries, with rates from 6% to 50% depending on the distance between the barcode and the effector molecule.

“barcode swapping can occur in retroviral vector libraries packaged as pools, and the degree of swapping can range from 6% to 50%, depending on the distance between the barcode and effector molecule (i.e. gRNA, shRNA, or cDNA) (Hill et al., 2018; Sack et al., 2016).”

• cites Hill et al., 2018 sourceNot checked yet

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• cites Sack et al., 2016 sourceNot checked yet

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May combine two claims.

This direct frequency measurement allows precise accounting of the number of cells sampled per population and improves analysis.

“This enables precise consideration of the number of cells sampled in each population and informs analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Psmb8 expression has been reported to correlate positively with tumor-infiltrating lymphocyte abundance in breast cancer.

“its expression has been found to positively correlate with tumor-infiltrating lymphocyte abundance in breast cancer (Lee et al., 2018).”

• cites Lee et al., 2018Not checked yet

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May combine two claims.

Generating genome-wide Pro-Code/CRISPR libraries cannot be done with the relative ease of DNA-barcoded libraries, so Pro-Codes will primarily be used for focused screens targeting 100-500 genes.

“Though generating genome-wide Pro-Code/CRISPR libraries cannot be done at the relative ease of DNA barcoded libraries, the Pro-Code’s will primarily be for more focused screens; concentrating on specific pathways or gene classes, and targeting 100–500 genes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

After acquisition, CyTOF data were normalized with bead-based normalization using the CyTOF software.

“After acquisition, the data were normalized using bead-based normalization using the CyTOF software.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cas9 expression was verified by western blot with an anti-Cas9 antibody.

“Cas9 expression was confirmed by western blot using anti-Cas9 antibody (Millipore, clone 7A9).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The only known targets of Rtp4 are opioid receptors.

“The only defined targets of Rtp4 are opioid receptors (Decaillot et al., 2008)”

• cites Decaillot et al., 2008 sourceNot checked yet

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Classified as probably not a substantial claim.

To ensure most transduced cells received only one vector, fewer than 10% of cells were transduced in all experiments.

“To ensure that a majority of transduced cells received only one vector, fewer than 10% of cells were transduced in all experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee reviewed and approved all animal protocols in the study.

“The Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai reviewed and approved all protocols for animal usage described in the present study.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Rtp4 is a chaperone protein that helps fold G protein coupled receptors.

“Rtp4 (Receptor transporter protein 4) is a chaperone protein involved in folding G protein coupled receptors (Decaillot et al., 2008).”

• cites Decaillot et al., 2008 sourceNot checked yet

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Classified as probably not a substantial claim.

Data were gated to remove normalization beads, debris, dead cells, and doublets, leaving NGFR+ events for clustering and high-dimensional analysis.

“The data were gated to exclude residual normalization beads, debris, dead cells and doublets, leaving NGFR+ events for clustering and high dimensional analyses.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CyTOF data were first debarcoded with Single Cell Debarcoder using post-assignment stringency filtering and outlier trimming.

“CyTOF data was first debarcoded using Single Cell Debarcoder (Zunder et al., 2015) using post-assignment debarcode stringency filter and outlier trimming.”

• cites Zunder et al., 2015 sourceNot checked yet

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