1. CRISPR Genome Editing Technologies
The continuous development of CRISPR-Cas genome editing tools is driving significant advances in the life sciences. Currently, four major classes of CRISPR-Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases, and prime editors—enable precise genome modifications in experimental systems, with some rapidly advancing into clinical applications. Each class exhibits unique capabilities and constraints. Our lab has contributed to expanding their editing potential, broadening their target range, and enhancing editing specificity.
2. in vivo CRISPR off-target analysis
CRISPR-based genome editing holds great promise for therapeutic applications. Before genome-editing tools can be applied in patients for disease mitigation, an assessment and optimization of their genomic safety is required, with the goal of minimizing unintended editing at off-target sites within the genome. We developed PE-tag technology and GUIDE-tag technology for identification of toxicity and specificity of CRISPR-based genomic medicine. GUIDE-tag enables one-step, unbiased off-target genome editing analysis in mouse liver and lung. Importantly, GUIDE-tag enables oncogene insertion to mouse lung or liver to build mouse models, which will facilitate cancer research. PE-tag enables genome-wide profiling of off-targets from prime editing in vitro using extracted genomic DNA, in mammalian cultured cell lines and in vivo in adult mouse liver. The PE-tag also offers the opportunity to perform off-target analysis directly on patient samples, providing a personalized assessment of genome-wide specificity. In summary, our off-target detection approaches provide platforms for the characterization and optimization of the specificity of CRISPR system in clinical trials.
a. Liang, S.Q., Liu, P., Ponnienselvan, K., Suresh, S., Chen, Z., Kramme, C., Chatterjee, P., Zhu, L.J., Sontheimer, E.J., Xue, W., and Wolfe, S.A. (2023). Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag. Nature Methods 20, 898-907.
b. Liang, S.Q., Liu, P., Smith, J.L., Mintzer, E., Maitland, S., Dong, X., Yang, Q., Lee, J., Haynes, C.M., Zhu, L.J., Watts, J.K., Sontheimer, E.J., Wolfe, S.A., and Xue, W. (2022). Genome-wide detection of CRISPR editing in vivo using GUIDE-tag. Nature Communications 13, 437.
c. Liang, S.Q., and Wolfe, S.A. (2023). Capturing prime editor off-target sites within the genome. Nature Methods 20, 801-802.
3. in vivo CRISPR delivery using AAV and LNP
Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. We developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. We observed ∼19%–26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. We further showed that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of angiotensin-converting enzyme 2 (ACE2) in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. In addition, leveraging a high-throughput platform, we synthesize and screen a combinatorial library of biodegradable ionizable lipids to build inhalable delivery vehicles for messenger RNA and CRISPR–Cas9 gene editors. Lead lipid nanoparticles are amenable for repeated intratracheal dosing and could achieve efficient gene editing in lung epithelium, providing avenues for gene therapy of congenital lung diseases. These highly effective delivery platforms will facilitate the study of therapeutic genome editing in the lung and other tissue types.
a. Liang, S.Q., Walkey, C.J., Martinez, A.E., Su, Q., Dickinson, M.E., Wang, D., Lagor, W.R., Heaney, J.D., Gao, G., and Xue, W. (2022b). AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway. Mol Ther 30, 238-243.
b. Liang, S.Q., Navia, A.W., Ramseier, M., Zhou, X., Martinez, M., Lee, C., Zhou, C., Wu, J., Xie, J., Su, Q., Wang, D., Flotte, T.R., Anderson, D.G., Tarantal, A.F., Shalek, A.K., Gao, G., and Xue, W. (2024). AAV5 Delivery of CRISPR/Cas9 Mediates Genome Editing in the Lungs of Young Rhesus Monkeys. Hum Gene Ther.
c. Li, B.*, Manan, R.S.*, Liang, S.Q.*, Gordon, A., Jiang, A., Varley, A., Gao, G., Langer, R., Xue, W., and Anderson, D. (2023). Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nature Biotechnology 41, 1410-1415.
4. Pathogenic allele correction and cancer modelling
using prime editor
Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. However, the efficacy of prime editing in adult mice has not been established. We developed an NLS-optimized prime editor that improves genome editing efficiency. Using those platforms, we could seed tumor formation through somatic cell editing in the adult mouse. We successfully utilized dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and our findings further established the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction. Delivery those platforms with two adeno-associated virus (AAV) vectors, we could correct the disease-causing mutation in a mouse model of type I tyrosinemia, and could seed tumor formation through somatic cell editing in the adult mouse. Our findings further established the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for cancer modeling and disease correction.
a. Liu, P.*, Liang, S.Q.*, Zheng, C., Mintzer, E., Zhao, Y.G., Ponnienselvan, K., Mir, A., Sontheimer, E.J., Gao, G., Flotte, T.R., Wolfe, S.A., and Xue, W. (2021). Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nature Commun 12, 2121.
b. Liang, S.Q., Liu, P., Xue, W., and Wolfe, S.A. (2023). Prime editor system for in vivo genome editing. US Patent App. 17/909,264
5. Identify drug resistant mechanisms in lung cancer using CRISPR screens
Oncogenic activation of KRAS and the FGFR amplification is frequent in lung and other cancers. However, due to drug resistance, pharmacological blockage of aberrant KRAS or FGFR signaling has provided little clinical benefit in patients. The determining factors for the limited efficacy remain incompletely understood. To identify genes that modulate sensitivity to targeted therapy, we performed kinome-scale CRISPR-Cas9 loss-of-function screens in KRAS-mutant lung cancer cell lines, FGFGR1-amplified squamous cell lines and malignant pleural mesothelioma cell lines. We identified PLK1 as a potent synthetic lethal target that mediates a resistance mechanism by overriding DNA damage and cell cycle arrest upon FGFR1 inhibition. We found that hyperactive mTOR signaling is a characteristic feature of chemoresistance in KRAS-mutant lung cancers. Coherently, combined treatment with mTOR inhibition and chemotherapy synergistically suppressed tumor growth in preclinical mouse models.
a. Liang, S.Q., Buhrer, E.D., Berezowska, S., Marti, T.M., Xu, D., Froment, L., Yang, H., Hall, S.R.R., Vassella, E., Yang, Z., Kocher, G.J., Amrein, M.A., Riether, C., Ochsenbein, A.F., Schmid, R.A., and Peng, R.W. (2019a). mTOR mediates a mechanism of resistance to chemotherapy and defines a rational combination strategy to treat KRAS-mutant lung cancer. Oncogene 38, 622-636.
b. Liang, S.Q., Marti, T.M., Dorn, P., Froment, L., Hall, S.R., Berezowska, S., Kocher, G., Schmid, R.A., and Peng, R.W. (2015). Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer. Cell Death Dis 6, e1824.
c. Yang, Z.*, Liang, S.Q.*, Saliakoura, M., Yang, H., Vassella, E., Konstantinidou, G., Tschan, M., Hegedus, B., Zhao, L., Gao, Y., Xu, D., Deng, H., Marti, T.M., Kocher, G.J., Wang, W., Schmid, R.A., and Peng, R.W. (2021a). Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer. EMBO Mol Med 13, e13193. (* Co-First Author)
d. Yang, Z.*, Liang, S.Q.*, Yang, H., Xu, D., Bruggmann, R., Gao, Y., Deng, H., Berezowska, S., Hall, S.R.R., Marti, T.M., Kocher, G.J., Zhou, Q., Schmid, R.A., and Peng, R.W. (2021b). CRISPR-Mediated Kinome Editing Prioritizes a Synergistic Combination Therapy for FGFR1-Amplified Lung Cancer. Cancer Res 81, 3121-3133. (* Co-First Author)
