Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

Abstract

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for ‘CRISPR for long-fragment integration via pseudovirus’), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and ‘cut sites’ to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

Main

Maximizing the therapeutic potential and improving the efficacy and safety of engineered cells often require the expression of large genes or of complex gene circuits¹. For example, chimeric antigen receptor (CAR) T cells leverage a single engineered receptor to rewire its cytotoxicity towards cancers^2,3. Efforts to improve their functionality, reduce toxicity or move beyond blood cancer into solid tumours, ageing, autoimmunity and viral clearance have leveraged combinations of tools that increase the ability of T cells to sense, process and respond to the disease. Biomolecular tools such as next-generation receptors^4,5,6, clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) and CRISPR interference (CRISPRi)^7,8,9,10, and logic gates^{Reporting summary}

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Extended data

Extended Data Fig. 1 CLIP’s cut sites enable more efficient HDR than IDLV.

CLIP strategy compared to IDLV (same as CLIP but without inclusion of cut sites) with and without RNP 3, 5, and 7 days after electroporation in K562s. The high expression peak is indicative of a targeted knock-in while the low expression peak is indicative of transient episomal expression of the reverse transcribed viral genome. Data from two biological replicates each with technical duplicates (replicate 1 shown in Fig. 2c).

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Extended Data Fig. 2 CLIP is robust to protocol perturbation in primary T cells.

a, CLIP pseudovirus transduced into primary T cells 48 hours or 24 hours before electroporation of RNP showed minimal effect on transduction efficiency. b, 2.5×10⁵, 5×10⁵, or 1×10⁶ cells were aliquoted into a cuvette for electroporation of Cas9-RNP, which had minimal effect on the knock-in efficiency. Data from one donor with technical duplicate.

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Extended Data Fig. 3 Pseudovirus delivers genetic payloads with low toxicity to primary T cells.

a, Viability of plasmid-based and PCR-based DNA donor knock-ins compared to CLIP at Day 4 in X-VIVO 15 media instead of RPMI 1640 (used in Figs. 4, 5, and 6). b, IDLV based donors (without cut sites) are also non-toxic to primary T cells. Data is from two donors each with technical replicates.

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Extended Data Fig. 4 CLIP integrates Cas13d into both ACTB and IL2RG, but is more highly expressed in ACTB.

a, Expression of Cas13d in either the ACTB or IL2RG locus in K562s (2 biological replicates with technical duplicates). b, Apparent knock-in efficiency is lower in IL2RG than ACTB (left) and the magnitude of expression of Cas13 is also lower in IL2RG than ACTB (right) (****: p < 0.0001) c, gDNA from K562s that underwent Cas13d CLIP in both loci was isolated. PCR was performed using a forward primer targeting Cas13d and reverse targeting IL2RG downstream of homology arm (left, expected band: 1101 bp) or using a forward primer targeting Cas13d and reverse targeting ACTB downstream of homology arm (right, expected band: 1059 bp). Data is from two biological replicates each with two technical replicates.

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Extended Data Fig. 5 CLIP enables Cas13d stabilization in primary T cells.

a, Magnitude of expression at Day 7 when GFP was knocked into IL2RG or ACTB in primary T cells (****: p < 0.0001). Histogram data shown in Figs. 4c,d and supplemental Fig. 6. b, Stability of mCherry-P2A-Cas13d in primary T cells compared between CLIP in the ACTB locus and randomly integrating lentiviruses driven by an EF1α or SFFV promoter (error bars represent SEM).

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Extended Data Fig. 6 hyperdCas12a-miniVPR expression when transfected but not transduced into HEK293T cells.

a, A representative histogram of the hyperdCas12-miniVPR-T2A-GFP expression in HEK293T cells 2 days after transfection. b, The MFI of the hyperdCas12a-miniVPR⁺ cells. c, When the same vector is packaged into lentivirus and transduced into K562 cells, no expression is seen 2 days after transduction.

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Extended Data Fig. 7 CLIP enables the expression and presentation of SARS-CoV-2 antigens.

a, Stability of S1-RdRP⁺ for both CLIP and EF1α driven lentivirus expression (error bars represent SEM). b, Histogram of sorted S1-RdRP⁺ Jurkat cells immediately before cryopreservation for immunopeptidomics.

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Source Data for Figs. 1–6 and Extended Data Figs. 1–7

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