CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling

Mou, Haiwei; Özata, Deniz M.; Smith, Jordan L.; Sheel, Ankur; Kwan, Suet‐Yan; Hough, Soren; Küçükural, Alper; Kennedy, Zachary; Cao, Yusong; Xue, Wen

doi:10.1186/s13073-019-0627-9

Cited by 15 publications

(19 citation statements)

References 41 publications

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“…We first tested whether biotin-dsDNA donors enable targeted NHEJ-mediated insertion in an easy-to-transfect mouse neuroblastoma cell line, N2A. We chose to insert an IRES-GFP fragment (1.7 Kb) into the 3′ UTR of Actb (beta-actin) at a previously described SpyCas9 target site, such that the insertion in the forward orientation leads to coexpression of GFP from the Actb promoter 41 (Fig. 1a, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1a, b, and Supplementary Table 1). Using one-step PCR with either unmodified primers or 5′ biotinylated primers from a plasmid donor template 32,41 , we generated unmodified linear dsDNA or biotin-dsDNA donor cassette, respectively (Supplementary Table 2). We transfected either dsDNA or biotin-dsDNA with or without expression plasmids for SpyCas9-mSA 34 (or SpyCas9) and sgRNA into N2A cells.…”

Section: Resultsmentioning

confidence: 99%

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Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

et al. 2022

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Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

et al. 2022

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“…In this approach gene integration was achieved by a dual sgRNA approach: one sgRNA linearized the donor plasmid while the second one targeted the integration site on the genome, the 3'-UTR region of the strongly expressed b-Actin gene. Even if this method has shown some success in tumor induction, it is however limited by events such as random integration of the plasmid donor, misorientation of the construct and inconsistency in the tumor penetrance (30).…”

Section: Livermentioning

confidence: 99%

SEMMs: Somatically Engineered Mouse Models. A New Tool for In Vivo Disease Modeling for Basic and Translational Research

Lima¹,

Maddalo

2021

Front. Oncol.

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Most experimental oncology therapies fail during clinical development despite years of preclinical testing rationalizing their use. This begs the question of whether the current preclinical models used for evaluating oncology therapies adequately capture patient heterogeneity and response to therapy. Most of the preclinical work is based on xenograft models where tumor mis-location and the lack of the immune system represent a major limitation for the translatability of many observations from preclinical models to patients. Genetically engineered mouse models (GEMMs) hold great potential to recapitulate more accurately disease models but their cost and complexity have stymied their widespread adoption in discovery, early or late drug screening programs. Recent advancements in genome editing technology made possible by the discovery and development of the CRISPR/Cas9 system has opened the opportunity of generating disease-relevant animal models by direct mutation of somatic cell genomes in an organ or tissue compartment of interest. The advent of CRISPR/Cas9 has not only aided in the production of conventional GEMMs but has also enabled the bypassing of the construction of these costly strains. In this review, we describe the Somatically Engineered Mouse Models (SEMMs) as a new category of models where a specific oncogenic signature is introduced in somatic cells of an intended organ in a post-natal animal. In addition, SEMMs represent a novel platform to perform in vivo functional genomics studies, here defined as DIVoS (Direct In Vivo Screening).

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“…Some tools are available that consider peptide cleaving and processing of proteins by the immunoproteasome, including NetChop20S, NetChopCterm, and ProteaSMM for HLA-I presentation and PepCleaveCD4 and MHC NP II for HLA-II presentation. Different methods that take aspects of peptide loading into account, for example affinity of the antigen-derived peptide for the transporter of antigen processing, are also in development [ 169 ]. In general, an optimal prediction tool is likely to arise from continued improvement and revision of existing prediction algorithms.…”

Section: Future Perspectivesmentioning

confidence: 99%

Neo-Antigen mRNA Vaccines

et al. 2020

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The interest in therapeutic cancer vaccines has caught enormous attention in recent years due to several breakthroughs in cancer research, among which the finding that successful checkpoint blockade treatments reinvigorate neo-antigen-specific T cells and that successful adoptive cell therapies are directed towards neo-antigens. Neo-antigens are cancer-specific antigens, which develop from somatic mutations in the cancer cell genome that can be highly immunogenic and are not subjected to central tolerance. As the majority of neo-antigens are unique to each patient’s cancer, a vaccine technology that is flexible and potent is required to develop personalized neo-antigen vaccines. In vitro transcribed mRNA is such a technology platform and has been evaluated for delivery of neo-antigens to professional antigen-presenting cells both ex vivo and in vivo. In addition, strategies that support the activity of T cells in the tumor microenvironment have been developed. These represent a unique opportunity to ensure durable T cell activity upon vaccination. Here, we comprehensively review recent progress in mRNA-based neo-antigen vaccines, summarizing critical milestones that made it possible to bring the promise of therapeutic cancer vaccines within reach.

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CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling

Cited by 15 publications

References 41 publications

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

SEMMs: Somatically Engineered Mouse Models. A New Tool for In Vivo Disease Modeling for Basic and Translational Research

Neo-Antigen mRNA Vaccines

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