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

Nature Biomedical Engineering volume 7, pages 661–671 (2023)Cite this article

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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.

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Fig. 1: Schematic design of CLIP.
Fig. 2: Engineering CLIP to enhance HDR in K562 cells.
Fig. 3: CLIP enables the simultaneous knock-ins of multiple payloads.
Fig. 4: CLIP is a non-toxic method for targeted essential gene knock-ins in human primary T cells.
Fig. 5: CLIP enables knock-in of large CRISPR–Cas payloads for the stable expression in K562 and primary T cells.
Fig. 6: CLIP enables proof-of-principle engineered cell vaccines with knock-in of multiple SARS-CoV-2 derived genes for antigen display.

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Data availability

The data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed immunopeptidome datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request. Key constructs and plasmids will be available on Addgene (https://www.addgene.org/Stanley_Qi/). Source data are provided with this paper.

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Acknowledgements

We thank V. Wiebking and M. Proteus for providing IL2RG knock-in plasmids. We thank Stanford Blood Center for providing peripheral blood mononuclear cells for isolating primary T cells. We thank Cayman Chemicals for performing MHC-I immunopeptidomics. We thank V. Ramani for help designing off-target experiments. M.C. acknowledges support from NSF GRFP, ARCS Foundation, NIH F31 fellowships and Siebel Scholarship. L.S.Q. acknowledges support from Li Ka Shing Foundation, National Science Foundation CAREER award (award number 2046650), Stanford Maternal & Child Health Research Institute (MCHRI) and California Institute for Regenerative Medicine (CIRM, DISC2-12669). The work is supported by a Li Ka Shing Foundation gift fund, National Science Foundation CAREER award (award number 2046650) and Stanford Maternal & Child Health Research Institute (MCHRI). L.S.Q. is a Chan Zuckerberg Biohub – San Francisco Investigator.

Author information

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Michael Chavez, Draven A. Rane, Xinyi Chen & Lei S. Qi

  2. Sarafan ChEM-H, Stanford University, Stanford, CA, USA

    Lei S. Qi

  3. Chan Zuckerberg Biohub—San Francisco, San Francisco, CA, USA

    Lei S. Qi

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  1. Michael Chavez
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Contributions

M.C. and L.S.Q. conceived of the idea and designed the experiments. M.C., D.A.R. and X.C. performed the experiments. M.C. and L.S.Q. analysed the experimental data. M.C. wrote the manuscript with input from L.S.Q. All authors read and approved the manuscript.

Corresponding author

Correspondence to Lei S. Qi.

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Competing interests

The authors have filed a related patent via Stanford University PCT/US2021/072335. L.S.Q. is a founder of Epic Bio and a scientific advisor of Kytopen, and Laboratory of Genomics Research. M.C. is a co-founder of Enoda Cellworks Inc. The other authors declare no competing interests.

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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).

Source data

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.

Source data

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.

Source data

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.

Source data

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).

Source data

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.

Source data

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.

Source data

Supplementary information

Supplementary Information

Supplementary figures, and nucleic acid and protein sequences.

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Chavez, M., Rane, D.A., Chen, X. et al. Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus. Nat. Biomed. Eng 7, 661–671 (2023). https://doi.org/10.1038/s41551-023-01037-x

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