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. 2018 May 30;140(21):6596-6603.
doi: 10.1021/jacs.8b01551. Epub 2018 May 18.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

Romain Rouet, Benjamin A Thuma  1 , Marc D Roy  2 , Nathanael G Lintner  3 , David M Rubitski  2 , James E Finley  2 , Hanna M Wisniewska  1 , Rima Mendonsa, Ariana Hirsh, Lorena de Oñate, Joan Compte Barrón, Thomas J McLellan  1 , Justin Bellenger  1 , Xidong Feng  1 , Alison Varghese  1 , Boris A Chrunyk  1 , Kris Borzilleri  1 , Kevin D Hesp  1 , Kaihong Zhou, Nannan Ma, Meihua Tu  3 , Robert Dullea  4 , Kim F McClure  3 , Ross C Wilson, Spiros Liras  3 , Vincent Mascitti  1 , Jennifer A Doudna  5
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Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

Romain Rouet et al. J Am Chem Soc. .
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Abstract

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Conflict of interest statement

Notes

The authors declare the following competing financial interest(s): S.L., V.M., B.A.T., J.A.D., and R.R. have filed intellectual property protection on tissue-specific genome engineering using CRISPR-Cas9 (U.S. Patent Application No. 62/254,652; WO2017/083368). J.A.D. is employed by HHMI and works at the University at California, Berkeley. UC Berkeley and HHMI have patents pending for CRISPR technologies on which she is an inventor. J.A.D. is the executive director of the Innovative Genomics Institute at UC Berkeley and UCSF. J.A.D. is a co-founder of Editas Medicine, Intellia Therapeutics, Mammoth Biosciences, Caribou Biosciences, and Scribe Therapeutics; a scientific advisor to Caribou, Intellia, Scribe, eFFECTOR Therapeutics, Inari, Synthego, and Metagenomi; and a board member of Driver and Johnson & Johnson. All authors, except Barrón, de Oñate, Doudna, Hirsh, Ma, Mendonsa, Rouet, Wilson, and Zhou, were employed by Pfizer, Inc. at the time this work was done.

Figures

Figure 1
(A) Receptor-facilitated, cell-selective gene editing. Schematic representation of Cas9-ASGPrL RNP and its transit from the extracellular medium to the nucleus. (B) Pyridyl disulfide ASGPr ligand and/or fluorophore precursors 1–3 and competing ligand 4 used in this study. (C) Legend of constructs used in this study, with corresponding ASGPr-binding affinities for various RNPs measured using SPR; reported values are from three replicates (standard error is reported); n.b.: no binding (top concentration tested = 10 nM). RNP made using sgRNA targeting EMX1.
Figure 2
Internalization in HEPG2 cells (ASGPr+) of Cas9-2lig-mCh (A) and Cas9-mCh (B) RNPs observed by live cell imaging at 1.5, 4, and 20 h; 20 h image contrast was adjusted down for clarity. Blue: Hoechst stain of cell nuclei. Green: Endolysosomal compartment stained using dextran488. Red: Intracellular Cas9 visualized via mCherry fluorescence. (C) Quantification of intracellular RNP accumulation in HEPG2 and SKHEP cells over 20 h. (D) Ligand competition experiment in HEPG2 cells with Cas9-2lig-mCh RNP and competing ASGPr ligand 4 (see the Supporting Information for more details). Fluorescence intensity was quantified using the sum of spots per cell (mean per well), reported as AU (absorbance units). Each data point (C,D) represents three technical replicate wells with a minimum of 10000 cells quantified per well. For (C) and (D), arithmetic means and standard deviations of the mean were calculated and plotted using GraphPad Prism version 7.02. Corresponding RNPs made from sgRNA targeting EMX1.
Figure 3
Internalization in HEPG2 cells (ASGPr+) of Cas9-2lig-AFr-1NLS (A) and Cas9-AFr-1NLS (B) RNPs observed by live cell imaging at 1.5, 4, and 20 h; 20 h images contrast was adjusted down for clarity. Blue: Hoechst stain of cell nuclei. Green: Endolysosomal compartment stained using dextran488. Red: Intracellular Cas9 visualized via AF647 fluorescence. (C) Quantification of intracellular RNP accumulation in HEPG2 cells over 20 h. (D) Quantification of intracellular RNP accumulation in SKHEP cells over 20 h. Fluorescence intensity was quantified using the sum of spots per cell (mean per well), reported as kA.U (103 absorbance units). Each data point (C, D) represents three technical replicate wells, with a minimum of 10000 cells quantified per well. For (C) and (D), arithmetic means and standard deviations of the mean were calculated and plotted using GraphPad Prism version 7.02. Corresponding RNPs made from sgRNA targeting EMX1.
Figure 4
Receptor-mediated uptake and genome editing. (A) Ligand competition experiment in HEPG2 cells (ASGPr+) with Cas9-2lig-AFr-1NLS RNP or Cas9-AFr-1NLS RNP using competing ASGPr ligand 4 (see the Supporting Information for more details). Fluorescence intensity was quantified using the sum of spots per cell (mean per well), reported as kA.U (103 absorbance units). Each data point represents three technical replicate wells, with a minimum of 10000 cells quantified per well. Arithmetic means and standard deviations of the mean were calculated and plotted using GraphPad Prism version 7.02. Corresponding RNPs made from sgRNA targeting EMX1. (B) Receptor-facilitated gene editing with Cas9-2lig-1NLS vs Cas9-1NLS RNP. Percentage indel rates derived from deep sequencing (n = 7–10 replicates; see also Table S1). Blue points represent samples treated with Cas9-2lig-1NLS, red points represent samples treated with Cas9–1NLS and green represents untreated controls. Diamonds represent assays done at Pfizer (Groton, CT) and circles represent assays done at UC Berkeley. The midpoint bars depict the geometric mean and the error bars depict the geometric standard deviation. The image was generated using Graphpad Prism© version 7.02. The corresponding RNPs were made from sgRNA targeting EMX1.
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Comment in

  • Targeting Cas9.
    Rusk N. Rusk N. Nat Methods. 2018 Jul;15(7):481. doi: 10.1038/s41592-018-0061-8. Nat Methods. 2018. PMID: 29967497 No abstract available.

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