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Efficient immune cell genome engineering with improved CRISPR editing tools

Waipan Chan, R. A. Gottschalk, Y. Yao, J. Pomerantz, R. N. Germain

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Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest. All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

In-depth summary

Claude Sonnet 4.6 · max effort · 8 Nov 2026 · from the full text of version 1 (15 Sept 2019) · CC BY 4.0

Doxycycline-inducible dual-color Cas9, purified lentivirus, puromycin-selected nickase HDR, integrase-deficient lentiviral HDR delivery and an all-in-one CRISPRa vector enable editing of macrophage and Jurkat T cells.

Macrophages are hurt by crude viral supernatant and nucleic acid sensing, and lymphocytes resist homology-directed repair (HDR) because transient delivery is toxic or inefficient and large-insert HDR is rare. This work combines inducible editing, cleaner lentivirus, selection-enriched nickase knock-in, and non-integrating lentiviral delivery of Cas9, gRNA and donor.

Key results

  1. ShownA doxycycline-inducible Cas9-2A-EGFP system plus a TagBFP gRNA vector gave strong DUSP3 knockout in RAW264.7 macrophages, and sequencing indicated that most targeted DUSP alleles were mutated.

    More than 95% reduction in DUSP3 protein in EGFP+ tBFP+ cells after 6 days at 1.0 µg/mL doxycycline (saturating dose); sequencing of 20 clones per gene across five DUSP genes; representative of three independent experiments. · Dual-color Inducible CRISPR-Cas9 Editing System…; Genomic DNA extraction, T7EN assay and…; Figure legends

  2. ShownPurified, concentrated lentivirus improved BMDM survival. DUSP1 gRNA gave sustained TLR4-induced p-p38 in BMDM but no detectable change in RAW cells with the same gRNA, indicating different MAPK regulation between the two cell types.

    Sustained p-p38 in LGB-DUSP1 BMDM vs empty-vector and DUSP4 controls (two independent experiments); the same DUSP1 gRNA gave no sustained p38 phosphorylation in RAW cells. No effect sizes reported. · Dual-color Inducible CRISPR-Cas9 Editing System…; Discussion; Figure legends

  3. ShownPuromycin selection with an in-frame PuroR-T2A-mNeonGreen donor and Cas9 nickase pairs enriched rare STAT3 knock-in Jurkat clones. CRE excision of the cassette allowed a second round of editing.

    6 of 7 mNG-medium/high clones were complete STAT3 knock-ins; 82.5% of clones partial or complete knock-in; co-transfection under 0.5%; more than 75% of mNG-BCL10+ cells gained mScarlet-STAT3 in round two; puromycin killed approximately 99.8% of NIL-CRE-treated cells. · An Efficient Selection Strategy for…

  4. SuggestedIntegrase-deficient lentiviral (NIL) delivery of Cas9, gRNA and HDR donor produced targeted knock-ins in Jurkat cells (CD3E mCherry, RelA tBFP) with functional RelA translocation. An overall knock-in rate is not reported in the text.

    Approximately 85% of tBFP-RelA cells showed nuclear translocation after PMA-ionomycin; approximately 70% lost tBFP after RelA gRNA knockout; NIL-LCv2R transduced more than 80% of human PBMC; CD3E and RelA flow data from two independent experiments. · All-in-one Non-integrating Lentiviral Delivery…; Discussion; Figure legends

  5. SuggestedA single-vector CRISPRa system (LentiSAMPHv2) induced PDCD1 or PDL1 surface expression in Jurkat cells at varying levels depending on the gRNA. A specificity check used cross-measurement of the other gene.

    6 gRNAs from an existing CRISPRa library tested against a no-gRNA control by flow cytometry; PDL1 measured for the top PD1-inducing gRNA. No numeric induction values given in the text. · Selective Gene Expression Induction with…; Figure legends

Methods, data and reproducibility

Lentiviral vectors for inducible Cas9-EGFP, fluorescent gRNA, Cas9 nickase HDR with puromycin selection and CRE excision, integrase-deficient all-in-one HDR delivery, and CRISPRa were built and tested by flow cytometry, T7EN assay, genomic PCR/sequencing and confocal imaging.

  • RAW264.7 macrophage line with inducible Cas9-2A-EGFP (RAWiCE); five DUSP genes targeted · mouse · n = 20 clones sequenced per gene
  • Bone marrow derived macrophages from C57BL/6 and Rosa26-Cas9 B6J mice · mouse
  • Jurkat T cells for STAT3, BCL10, CD3E and RelA knock-in and PDCD1/PDL1 CRISPRa · human · n = 40 puromycin-resistant STAT3 clones; 7 analyzed by genomic PCR
  • Human PBMC from healthy anonymous donors transduced with NIL-LCv2R · human
Methods & tools
pCW-Cas9 and pCW-Cas9-2A-EGFP (iCE), lentiCRISPRv2 derivatives (LGB, LGR, LCv2B, LCv2S, LCv2R, LCv2E), Cas9-D10A nickase (PX461, PX462), psPAX2-D64V integrase-deficient packaging, T7 Endonuclease I assay, crispr.mit.edu gRNA design, Gibson Assembly cloning, LentiSAMPHv2 CRISPRa, FlowJo, Imaris, Leica SP8 confocal microscopy
Reagents & environment
Flow cytometry data processed with FlowJo and imaging data with Imaris (Bitplane); no software versions stated.
Reproducibility gaps
Constructs are to be deposited with Addgene for qualified investigators; no raw data or code repository is stated.
Limitations (5)
  • The puromycin selection strategy needs a well-expressed target locus. It worked best with N-terminal PuroR-2A fusions, not C-terminal 2A-PuroR. · stated by the authors
  • T7EN assays do not give the true knockout percentage and antibody availability was limited, so DUSP editing outcomes rely partly on sequencing of 20 clones per gene. · stated by the authors
  • Inserting the PuroR cassette with a fluorescent protein can sharply reduce expression of a small protein such as BCL10 and impair its oligomerization, so the cassette must be removed. · stated by the authors
  • Residual random integration events from NIL particles remain a possible concern. The authors propose adding sensitive markers such as mScarlet or tEGFR in future vector designs to eliminate them. · stated by the authors
  • Most phenotypes come from immortalized lines (RAW264.7, Jurkat) with representative flow plots from two or three experiments and few reported statistics or replicate counts; CRISPRa data are qualitative. · noted in this summary

Read this if you need vector designs and selection strategies for CRISPR knockout, knock-in or activation in macrophages, BMDM or Jurkat T cells.

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