Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

Theodore L. Roth, P. Jonathan Li, Jasper F. Nies, Ruby Yu, Michelle L.T. Nguyen, Youjin Lee, Ryan Apathy, Anna Truong, Joseph Hiatt, David Wu, David N. Nguyen, Daniel Goodman, Jeffrey A. Bluestone, Kole Roybal, Eric Shifrut, Alexander Marson

Preprint posted on 12 April 2019

T-cells for everyone! A platform to screen functionality of novel DNA sequences in T-cells.

Selected by Pavithran Ravindran


The engineering of a patient’s immune cells to specifically target and kill cancer cells has become a field of research with a lot of momentum in recent years. Ever since the advent of genetically engineering a patient’s T cells to produce chimeric antigen receptors (CAR-T cells), which have even shown efficacy in clinical, a number of researchers have developed various synthetic receptors that extend this initial breakthrough1,2. For instance, one group developed a receptor that uses the immunosuppressive signal, TGFβ, and rewires the signal into an activating signal for T-cells3. Furthermore, a lot of beautiful work has been done to develop novel receptors that can be used to target specific combinations of cancer targets to reduce off-target effects4–6. Although this research has exploded in the past few years, it is clear that developing and validating novel DNA sequences for receptors is quite difficult and new methods to do so would help this field tremendously. The authors of this preprint attempt to do just this. They first develop a method to massively parallelize the insertion of novel DNA sequences into the genome of T-cells. They then use this to create a library of T-cells with novel receptors and put this library through a number of selection pressures to obtain known and novel DNA insert sequences. Finally, they even go as far as to take this library and use it in vivo with a tumor mouse model to identify possibly new mechanisms of helping T-cells navigate the tumor microenvironment.


Key Findings

In this preprint, the authors set out to develop a method to rapidly develop novel DNA sequences in T-cells that encode proteins to increase T-cell functionalities and allow them to combat the ever-evolving landscape of cancer. The authors had recently published a method that allows them to edit the genomes of T-cells using CRISPR-Cas9 technology with remarkable efficiency7. As a first step toward the goal of creating a screening platform for specific T-cell functions, the authors expanded their previous work to understand the breadth of genes that could be screened. They utilized an 800 base-pair reporter gene and CRISPR tagged this cassette to 91 genes and found that 88% of the loci were amenable to genomic integration of the reporter. Furthermore, they were able to analyze the efficiency for each gene and found that gRNA sequence, RNA expression of the target gene and chromatin accessibility were able to explain the variation. Next, they wanted to test the depth to which they could study novel sequences to reprogram T-cells. Specifically, they wanted to create targeted knock-in that integrate synthetic elements with existing sequences, Genetically Engineered Endogenous Proteins (GEEPs), by varying the gene regulation, gene product, or the intra/extracellular domains of endogenous T-cell receptors. As a proof of principle, the authors decided to engineer the IL2RA gene and, remarkably, they were able to increase expression of the receptor by knocking-in a constitutive promoter, and impart the stimulus dependent expression of the endogenous gene to a novel receptor by placing the new receptor in front of the endogenous protein with a self-cleaving peptide tag. Furthermore, they were able to completely replace entire domains of the gene. Overall, the authors undertook and accomplished the herculean task of CRISPR tagging 91 genes and then developing GEEPs with IL2RA.

Figure 1. Schematic of overall CRISPR/GEEP screen in T-cells. Adapted from figure 3 from original preprint.


With these results, the authors were situated to develop a screen in which they could screen through various polycistronic DNA sequences that modify T cell function for particular therapeutic contexts (Figure 1). As their test bed, the authors decided to modify the endogenous TRAC gene with new TCRβ and TCRα sequences alone with a novel receptor inserted in the middle along with a barcode sequence. This was then incorporated into T-cells and the library was tested for knock-in of the various inserts. To accomplish this, the authors introduced a small sequence of DNA in the homology arms that does not match the endogenous sequence far from the insert location. Based on the fact that with on-target HDR, integration does not incorporate the entire homology arm while template switching does, the authors could sequence this small sequence of DNA and if they got back the original sequence, then on-target HDR occurred, while if the new sequence was there, there may have been off-target integration. The authors knocked in 36 different inserts into T-cells containing dominant negative receptors, synthetic receptors, transcription factors and metabolic regulators. With this pooled library of T-cells, the authors then wanted to ask what DNA sequences would become enriched after anti-CD3/CD28 stimulation. The authors found a novel Fas “switch” receptor with a 4-1BB intracellular domain. This switch receptor takes in the input of FASL, which activates apoptosis in T-cells upon binding to Fas, and redirects it into a T-cell persistence output. This result was highly reproducible and highlights the versatility of this screening platform to identify novel knock-in synthetic receptors that promote specific phenotypes in particular environments.

The authors took this one more step further by applying their screen in an in vivo context. The authors introduced their T-cell library into a mouse bearing human melanoma expressing the NY-ESO-1 antigen. Upon extraction of T-cells from the tumor 5 days after in vivo tumor expansion, the authors identified a unique metabolic protein, MCT4, that had not shown enrichment in any of their other screens. They also found 3 strong hits (an anti-suppressive TGF R2-41BB chimera, an anti- apoptotic FAS-41BB chimera, and the transcriptional program altering TCF7) that they decided to do individual validation of. Remarkably, each were not only able to increase T-cells’ ability to grow but also to eliminate the melanoma cancer cells. Overall, this work enables the use of endogenous genome editing of T-cells to find novel DNA sequences that provide synthetic T-cells an advantage to kill cancer cells in a context dependent manner.


Why I chose this preprint

I chose this preprint simple due to the herculean effort that this work seem to encompass. Not only did the authors of this preprint make a protocol that allowed them to CRISPR tag almost 90 genes in T-cells, they also developed a way to make large insertions into T-cells, created a library of T-cells with endogenous and novel receptors, and screened the library both in in vitro and in vivo contexts. The paper was very easy to read and the figures clearly communicated the science being showcased. Overall, the authors managed to take very complex and massive amount of data and communicate it in a very digestible manner; thus, this paper was truly a joy to read.


Questions for the authors

  1. What is the biggest insert that you were able to do with your protocol and do you think this is the limit? Could one do not just a single novel insert but multiplex to understand the combinatorial effects of different receptors/TFs/metabolic regulators etc.? Better yet, could one screen for combinatorial gate logic (AND, ANDNOT, etc.) using this?
  2. How easily does your protocol transfer to different types of cells? Could you use this CRISPR protocol to modify multiple types of immune cells to understand their effects in combination with one another?



  1. Maude, S. L., Teachey, D. T., Porter, D. L. & Grupp, S. A. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood 125, 4017–4024 (2015).
  2. Milone, M. C. et al. Chimeric Receptors Containing CD137 Signal Transduction Domains Mediate Enhanced Survival of T Cells and Increased Antileukemic Efficacy In Vivo. Mol. Ther. 17, 1453–1464 (2009).
  3. Chang, Z. L. et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat. Chem. Biol. 14, 317–324 (2018).
  4. Roybal, K. T. et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 1–14 (2016).
  5. Roybal, K. T. et al. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell 164, 770–779 (2016).
  6. Cho, J. H. et al. Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses Article Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses. Cell 1–13 (2018). doi:10.1016/j.cell.2018.03.038
  7. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018).

Tags: crispr, immunotherapy

Posted on: 18 June 2019 , updated on: 19 June 2019


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