Pooled targeted knockin of long DNA sequences with single-cell RNA sequencing was developed.

  • Major Finding: Pooled targeted knockin of long DNA sequences with single-cell RNA sequencing was developed.

  • Concept: This method revealed enhanced antitumor efficacy of T cells with a TGFβR2–41BB chimeric receptor.

  • Impact: This proof-of-concept work shows that the technique can speed discovery in cellular immunotherapy.

Therapies based on adoptive transfer of immune cells, such as chimeric antigen receptor T-cell therapies, have shown great promise for treating some cancers. Efforts to enhance the efficacy of these treatments via genetic knockin are ongoing, but one limitation has been the inability to quickly screen large DNA knockins to determine their impact on antitumor efficacy. To address this, Roth and colleagues developed a technique that employs nonviral CRISPR targeting in human T cells to knock in pools of candidate genetic constructs and test their effects on T-cell function in vitro and in vivo in a high-throughput fashion. Applying this approach to insert a library of 36 barcoded long DNA sequences—encoding dominant-negative receptors, synthetic switch receptors with engineered intracellular domains, and heterologous transcription factors, metabolic regulators, and receptors—into the T-cell receptor locus enabled the identification of sequences that improved T cells’ fitness and ability to kill cancer cells in vitro. By combining the pooled knockin screening with single-cell RNA sequencing, the authors developed pooled knockin sequencing (dubbed PoKI-seq), which made it possible to phenotypically characterize individual cells harboring one or more knockin constructs. PoKI-seq experiments identified a novel TGFβR2–41BB chimeric receptor as being of interest, and in vivo experiments using mice bearing human melanoma xenografts demonstrated that T cells with this receptor knocked in had improved antitumor efficacy and increased abundance of tumor-infiltrating lymphocytes, which exhibited increased expression of genes encoding effector cytokines. Collectively, these experiments demonstrate the potential of PoKI-seq to aid discovery of DNA sequences that enhance the efficacy of cellular immunotherapies, increasing the rate of progress in this developing field.

Roth TL, Li PJ, Blaeschke F, Nies JF, Apathy R, Mowery C, et al. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell 2020 Apr 16 [Epub ahead of print].

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