TEG-Seq: An Ion Torrent-Adapted NGS Workflow for
            <i>in Cellulo</i>
            Mapping of CRISPR Specificity

Tang, Pei-Zhong; Bo, Ding; Peng, Lansha; Mozhayskiy, Vadim; Potter, Jason; Chesnut, Jonathan D.

doi:10.2144/btn-2018-0105

Cited by 10 publications

(8 citation statements)

References 31 publications

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“…Seventy-nine bioinformatically-predicted off-target sites for gRNA #1-#5 were evaluated by Targeted Amplicon-Seq, and no off-target activity was detected at any of the predicted sites for any of the gRNAs ( online supplemental table S5 ). TEG-seq 12 was used to further evaluate off-target activity throughout the genome for gRNA #3 and gRNA #4. Three potential off-target sites were identified for gRNA #3, and five potential off-target sites were identified for gRNA #4 ( online supplemental figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

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CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling

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Sakellariou-Thompson

et al. 2022

J Immunother Cancer

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BackgroundThe correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (NCT03610490). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR/Cas9-mediated knockout of the TGF-β receptor 2 (TGFBR2) in patient-derived OvCa TIL.MethodsOvCa TILs were generated from four patients’ tumor samples obtained at surgical resection and subjected to CRISPR/Cas9-mediated knockout of TGFBR2 before undergoing a rapid expansion protocol. TGFBR2-directed gRNAs were comprehensively evaluated for their TGFBR2 knockout efficiency and off-target activity. Furthermore, the impact of TGFBR2 knockout on TIL expansion, function, and downstream signaling was assayed.ResultsTGFBR2 knockout efficiencies ranging from 59±6% to 100%±0% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. TGFBR2 knockout TIL were resistant to immunosuppressive TGF-β signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-β stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-β, and improved cytotoxicity in the presence of TGF-β. CRISPR-modification itself did not alter the ex vivo expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two TGFBR2-targeting gRNAs.ConclusionsCRISPR/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific TGFBR2 knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-β. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well.

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Section: Resultsmentioning

confidence: 99%

“…Second, target-enriched GUIDE-seq (TEG-seq) 12 was used for genome-wide detection of off-target cleavage using gRNAs #3 and #4. For TEG-seq analysis, TIL were transfected with Cas9 RNP complexes with the addition of 20 pmol double stranded DNA tag to integrate into double strand break locations.…”

Section: Methodsmentioning

confidence: 99%

CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling

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Sakellariou-Thompson

et al. 2022

J Immunother Cancer

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“…For emerging of new data experimentally verified, firstly, we collected off-target sequences as well as their sgRNAs from in vitro [8]- [10], [22], [23] and cell-based genomewide assays [11]- [13], [24], among these assays, 3 of them contain PCR validation experiments [8], [22], [23]. As we hypothesized that the in vitro and cell-based assays may not be the reliable off-target data sources due to the differences in setting the environments between them and PCR assays, we further split the off-targets to two types.…”

Section: A Datasetsmentioning

confidence: 99%

“…In vitro methods can detect off-target sites with low mutation frequency, for example, Kim et al introduced Digenome-seq to profile off-target sites on whole genome sequencing [8], Tsai et al developed CIRCLE-seq by reducing random reads in Digenome-seq [9], and Cameron et al presented SITE-seq by enriching and tagging Cas9 cleavage sites in Digenome-seq [10]. Additionally, cell-based methods can identify the mutation in certain cell types and conditions, for instances, GUIDE-seq is proposed to find off-target sites by tagging DNA double-strand breaks (DSBs) with small double-stranded oligonucleotides [11], BLISS is developed to label the DSBs directly [12], and TEGseq is presented to enrich the target in GUIDE-seq [13].…”

Section: Introductionmentioning

confidence: 99%

DL-CRISPR: A Deep Learning Method for Off-Target Activity Prediction in CRISPR/Cas9 With Data Augmentation

et al. 2020

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is a popular and easy to use gene-editing technique, but it has off-target risk. Cutting the off-target sites will harm the cells severely, hence in silico methods are needed to help to avoid this. Most existing in silico approaches mainly relied on a relatively small positive dataset and the data imbalance issue still exists. Besides, some samples used to be considered as negative are later proved to be positive. Hence, it is essential to refresh the dataset and develop more accurate off-target activity prediction programs. In this work, firstly, we extended the current positive dataset and explored the potential differences between positive and negative data based on the new dataset. Then we adopted a new data augmentation method to solve the data imbalance issue, and used the ensemble idea to take more negative data into consideration to make the model close to the real scenario, but at the same time keeping the model balance. Finally, we developed DL-CRISPR, a deep learning framework to predict off-target activity in CRISPR/Cas9. DL-CRISPR is evaluated and compared with other state-of-the-art methods on three kinds of datasets: 5-fold cross validation test datasets, putative off-targets datasets related to specific single guide RNAs (sgRNAs), and putative off-targets datasets related to unseen sgRNAs. DL-CRISPR realizes the best average accuracy, i.e. 98.57%, on 5-fold cross validation datasets and correctly detects more off-targets on datasets related to both seen and unseen sgRNAs. INDEX TERMS CRISPR/Cas9, data augmentation, deep learning, off-target.

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“…The impact of CRISPR modification on TIL expansion and TCR clonal diversity was evaluated. Finally, the risk of off-target CRISPR activity throughout the genome was evaluated using Target Enriched GUIDE-seq (TEG-seq) 2 followed by next generation sequencing (NGS) validation of putative off-target sites. Results Using five TGFBR2-directed guide RNAs (gRNAs), we achieved gene disruption efficiencies ranging from 48%-90%, which correlated inversely with the degree of SMAD phosphorylation after TGF-b exposure (r=-0.9440, p=0.0158, n=4 donors) (figure 1A-C).…”

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confidence: 99%

172 Overcoming immunosuppressive TGF-β signaling in human ovarian cancer-derived tumor infiltrating lymphocytes via non-viral CRISPR engineering

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Sakellariou-Thompson

et al. 2021

Regular and Young Investigator Award Abstracts

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BackgroundOur ongoing clinical trial for the treatment of melanoma with TGF-β-resistant tumor-infiltrating lymphocytes (TIL) [TGF-β dominant negative receptor 2 (TGFβDNR2) transduced-TIL] has yielded long-term responses in checkpoint refractory patients (NCT01955460). Building on this success, we sought to extend the impact of TGF-β–resistant TIL therapy to additional cancers while optimizing a non-viral alternative to transduction with a TGFβDNR2. Ovarian cancer (OvCa), which is characterized by an abundance of TGF-β, a dysfunctional immune infiltrate, and a paucity of novel treatment options, is an ideal candidate for TGF-β–resistant TIL therapy. Here, we present an optimized and clinically-scalable method for CRISPR/Cas9-mediated deletion of the TGF-β receptor (TGFBR2) in OvCa TIL.MethodsOvCa TIL were generated from tumor fragments1 and subjected to CRISPR-mediated knockout of TGFBR2 before going through a rapid expansion protocol. Resistance of TGFBR2-knockout TIL to TGF-β signaling was evaluated via quantification of downstream SMAD-2/-3 phosphorylation, global transcriptional changes upon TGF-β exposure, and cytokine release following TCR stimulation in the presence of TGF-β. The impact of CRISPR modification on TIL expansion and TCR clonal diversity was evaluated. Finally, the risk of off-target CRISPR activity throughout the genome was evaluated using Target Enriched GUIDE-seq (TEG-seq)2 followed by next generation sequencing (NGS) validation of putative off-target sites.ResultsUsing five TGFBR2-directed guide RNAs (gRNAs), we achieved gene disruption efficiencies ranging from 48%–90%, which correlated inversely with the degree of SMAD phosphorylation after TGF-β exposure (r=-0.9440, p=0.0158, n=4 donors) (figure 1A-C). TGF-β exposure induced a strong transcriptional response in wild-type TIL but had little to no effect on TGFBR2-knockout TIL (figure 2). TGFBR2-knockout TIL functioned well in the presence of exogenous TGF-β as evidenced by equally strong secretion of pro-inflammatory cytokines in the presence and absence of TGF-β (figure 3). CRISPR-modification did not hamper the ex vivo expansion efficiency nor the TCR clonal diversity of expanded OvCa TIL (figure 4). Using TEG-seq, we identified ≤5 low-probability off-target sites for gRNA-#3 and gRNA-#4, each of which were attributed to background sequencing artifacts upon further validation by NGS of specific amplicons (figure 5).Abstract 172 Figure 1(A) Genomic-level TGFBR2 knockout efficiency using 5 different gRNAs as evidenced by NGS of specific amplicons (n=1 TIL donor). (B) SMAD-2 and SMAD-3 phosphorylation in TGFBR2 knockout TIL vs. control TIL after 30 min exposure to TGF-β1. The left panel shows representative histograms of phospho-SMAD staining, and the right panel shows quantification of cells positive for phospho-SMAD-2/-3 after TGF-β exposure (n=4 TIL donors). The statistical significance of each experimental condition compared to the non-transfected control is shown. (C) Inverse correlation of TGFBR2 knockout efficiency and TGF-β-mediated SMAD phosphorylation.Abstract 172 Figure 2Top 100 differentially expressed genes in non-transfected (WT) TIL exposed to TGF-β. TGFBR2 knockout (KO) TIL display minimal gene expression changes upon TGF-β exposure (n=3 technical replicates).Abstract 172 Figure 3TIL were collected after 14 days of expansion and re-stimulated with 300 ng/mL plate-bound anti-CD3 in the presence of 3000 IU/mL IL2 and 10 ng/mL human TGF-β1 or vehicle. Cell culture supernatant was collected after 72 hrs of stimulation and assayed for the presence of 10 proinflammatory cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α). For TIL with intact TGFBR2 (non-transfected and Cas9 mock transfected TIL), the production of many pro-inflammatory cytokines decreased significantly in the presence of TGF-β. Conversely, TGFBR2 knockout TIL (generated using gRNA #3 or gRNA #4) retain cytokine secretion in the presence of TGF-β. IL-12p70 was below the limit of detection in this assay and is therefore not presented.Abstract 172 Figure 4(A) Control and CRISPR-modified OvCa TIL expand with equal efficiency during a 14-day rapid expansion protocol. Fold expansions ranging from 1000× - 3000× were observed across 4 independent patient samples. (B) The TCR clonal diversity of TIL after 14-day expansion was assessed by TCRB sequencing. Productive Simpson Clonality was equivalent in CRISPR-modified TIL compared to control TIL samples.Abstract 172 Figure 5TEG-seq revealed 3 putative off-target sites for gRNA #3 and 5 putative off-target sites for gRNA #4. The aligned sequences show similarities and differences between the gRNA sequence and the reference genome site. Dots represent exact matches in the reference genome compared to the gRNA sequence. Dashes represent missing bases, lower-case letters represent extra bases, and upper-case letters represent a base mismatch. Validation by NGS of specific amplicons confirmed the presence of TEG-seq Tag integration and large indels at the on-target cleavage sites for gRNA #3 and #4, indicating successful Cas9 editing and Tag integration in our experiment. NGS validation revealed that all putative low probability off-target sites were background artifacts as evidenced by the lack of Tag identification and lack of large indels.ConclusionsCRISPR/Cas9-mediated knockout of TGFBR2 is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods that yield little to no evidence of off-target activity. This study lays the groundwork for clinical translation of CRISPR-modified, TGF-β-resistant TIL for OvCa treatment, which will not only provide a novel immunotherapy for OvCa patients but also a platform for engineering more potent TIL therapies in general.ReferencesSakellariou-Thompson D, Forget MA, Hinchcliff E, Celestino J, Hwu P, Jazaeri AA, et al. Potential clinical application of tumor-infiltrating lymphocyte therapy for ovarian epithelial cancer prior or post-resistance to chemotherapy. Cancer Immunology, Immunotherapy: CII 2019;68(11):1747–57.Tang PZ, Ding B, Peng L, Mozhayskiy V, Potter J, Chesnut JD. TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity. Bio Techniques 2018;65(5):259–67.Ethics ApprovalAll procedures performed were in accordance with the 1975 Helsinki declaration. Ethical approval and tissue from surgical resections used to expand TIL were both obtained under protocols (PA16-0912 and LAB02-188) approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center. Written informed consent was obtained from all individual participants included in the study for their specimens and data to be used in research and for publication.

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TEG-Seq: An Ion Torrent-Adapted NGS Workflow for in Cellulo Mapping of CRISPR Specificity

Cited by 10 publications

References 31 publications

CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling

CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling

DL-CRISPR: A Deep Learning Method for Off-Target Activity Prediction in CRISPR/Cas9 With Data Augmentation

172 Overcoming immunosuppressive TGF-β signaling in human ovarian cancer-derived tumor infiltrating lymphocytes via non-viral CRISPR engineering

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