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Volume 41 Issue 9, September 2023

Large-scale knock-in T cell engineering

Massively parallel knock-in engineering of primary human T cells with an AAV library. Dai et al. screen a large pool of differentially mutated CAR-T cells for genetic perturbations that enhance function.

See Dai et al.

Image: Xiaoyun Dai and Sidi Chen, Yale University. Cover Design: Erin Dewalt.

Volume 41 Issue 9

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  • CLASH is a CRISPR-based platform that enables the parallel knock-in via homology-directed repair of a large pool of transgene variants encoded in adeno-associated virus vectors. CLASH can be applied to the systematic and unbiased selection of favorable features in T cells and, in principle, other cell types.

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  • Targeted RNA degradation remains a challenge using existing methods owing to off-target effects and toxicity. We adapted the CRISPR-Csm complex, a multi-protein effector from type III CRISPR systems, for precise knockdown of nuclear or cytoplasmic transcripts in eukaryotic cells with minimal side effects.

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  • Ultrasensitive sequential fluorescence in situ hybridization (USeqFISH) enables multiplexed detection of the expression of endogenous and exogenous genes delivered by adeno-associated virus (AAV) vectors in intact tissue. USeqFISH provides a spatial map of AAV tropism with high throughput and resolution.

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