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      CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

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          Abstract

          A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

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          Sensitivity of Chest CT for COVID-19: Comparison to RT-PCR

          Summary In a series of 51 patients with chest CT and RT-PCR assay performed within 3 days, the sensitivity of CT for COVID-19 infection was 98% compared to RT-PCR sensitivity of 71% (p<.001). Introduction In December 2019, an outbreak of unexplained pneumonia in Wuhan [1] was caused by a new coronavirus infection named COVID-19 (Corona Virus Disease 2019). Noncontrast chest CT may be considered for early diagnosis of viral disease, although viral nucleic acid detection using real-time polymerase chain reaction (RT-PCR) remains the standard of reference. Chung et al. reported that chest CT may be negative for viral pneumonia of COVID-19 [2] at initial presentation (3/21 patients). Recently, Xie reported 5/167 (3%) patients who had negative RT-PCR for COVID-19 at initial presentation despite chest CT findings typical of viral pneumonia [3]. The purpose of this study was to compare the sensitivity of chest CT and viral nucleic acid assay at initial patient presentation. Materials and Methods The retrospective analysis was approved by institutional review board and patient consent was waived. Patients at Taizhou Enze Medical Center (Group) Enze Hospital were evaluated from January 19, 2020 to February 4, 2020. During this period, chest CT and RT-PCR (Shanghai ZJ Bio-Tech Co, Ltd, Shanghai, China) was performed for consecutive patients who presented with a history of 1) travel or residential history in Wuhan or local endemic areas or contact with individuals with individuals with fever or respiratory symptoms from these areas within 14 days and 2) had fever or acute respiratory symptoms of unknown cause. In the case of an initial negative RT-PCR test, repeat testing was performed at intervals of 1 day or more. Of these patients, we included all patients who had both noncontrast chest CT scan (slice thickness, 5mm) and RT-PCR testing within an interval of 3 days or less and who had an eventual confirmed diagnosis of COVID-19 infection by RT-PCR testing (Figure 1). Typical and atypical chest CT findings were recorded according to CT features previously described for COVD-19 (4,5). The detection rate of COVID-19 infection based on the initial chest CT and RT-PCR was compared. Statistical analysis was performed using McNemar Chi-squared test with significance at the p <.05 level. Figure 1: Flowchart for patient inclusion. Results 51 patients (29 men and 22 women) were included with median age of 45 (interquartile range, 39- 55) years. All patients had throat swab (45 patients) or sputum samples (6 patients) followed by one or more RT-PCR assays. The average time from initial disease onset to CT was 3 +/- 3 days; the average time from initial disease onset to RT-PCR testing was 3 +/- 3 days. 36/51 patients had initial positive RT-PCR for COVID-19. 12/51 patients had COVID-19 confirmed by two RT-PCR nucleic acid tests (1 to 2 days), 2 patients by three tests (2-5 days) and 1 patient by four tests (7 days) after initial onset. 50/51 (98%) patients had evidence of abnormal CT compatible with viral pneumonia at baseline while one patient had a normal CT. Of 50 patients with abnormal CT, 36 (72%) had typical CT manifestations (e.g. peripheral, subpleural ground glass opacities, often in the lower lobes (Figure 2) and 14 (28%) had atypical CT manifestations (Figure 3) [2]. In this patient sample, difference in detection rate for initial CT (50/51 [98%, 95% CI 90-100%]) patients was greater than first RT-PCR (36/51 [71%, 95%CI 56-83%]) patients (p<.001). Figure 2a: Examples of typical chest CT findings compatible with COVID-19 pneumonia in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 74 years old with fever and cough for 5 days. Axial chest CT shows bilateral subpleural ground glass opacities (GGO). B, female, 55 years old, with fever and cough for 7 days. Axial chest CT shows extensive bilateral ground glass opacities and consolidation; C, male, 43 years old, presenting with fever and cough for 1 week. Axial chest CT shows small bilateral areas of peripheral GGO with minimal consolidation; D, female, 43 years old presenting with fever with cough for 5 days. Axial chest CT shows a right lung region of peripheral consolidation. Figure 2b: Examples of typical chest CT findings compatible with COVID-19 pneumonia in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 74 years old with fever and cough for 5 days. Axial chest CT shows bilateral subpleural ground glass opacities (GGO). B, female, 55 years old, with fever and cough for 7 days. Axial chest CT shows extensive bilateral ground glass opacities and consolidation; C, male, 43 years old, presenting with fever and cough for 1 week. Axial chest CT shows small bilateral areas of peripheral GGO with minimal consolidation; D, female, 43 years old presenting with fever with cough for 5 days. Axial chest CT shows a right lung region of peripheral consolidation. Figure 2c: Examples of typical chest CT findings compatible with COVID-19 pneumonia in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 74 years old with fever and cough for 5 days. Axial chest CT shows bilateral subpleural ground glass opacities (GGO). B, female, 55 years old, with fever and cough for 7 days. Axial chest CT shows extensive bilateral ground glass opacities and consolidation; C, male, 43 years old, presenting with fever and cough for 1 week. Axial chest CT shows small bilateral areas of peripheral GGO with minimal consolidation; D, female, 43 years old presenting with fever with cough for 5 days. Axial chest CT shows a right lung region of peripheral consolidation. Figure 2d: Examples of typical chest CT findings compatible with COVID-19 pneumonia in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 74 years old with fever and cough for 5 days. Axial chest CT shows bilateral subpleural ground glass opacities (GGO). B, female, 55 years old, with fever and cough for 7 days. Axial chest CT shows extensive bilateral ground glass opacities and consolidation; C, male, 43 years old, presenting with fever and cough for 1 week. Axial chest CT shows small bilateral areas of peripheral GGO with minimal consolidation; D, female, 43 years old presenting with fever with cough for 5 days. Axial chest CT shows a right lung region of peripheral consolidation. Figure 3a: Examples of chest CT findings less commonly reported in COVID-19 infection (atypical) in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 36 years old with cough for 3 days. Axial chest CT shows a small focal and central ground glass opacity (GGO) in the right upper lobe; B, female, 40 years old. Axial chest CT shows small peripheral linear opacities bilaterally. C, male, 38 years old. Axial chest CT shows a GGO in the central left lower lobe; D, male, 31 years old with fever for 1 day. Axial chest CT shows a linear opacity in the left lower lateral mid lung. Figure 3b: Examples of chest CT findings less commonly reported in COVID-19 infection (atypical) in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 36 years old with cough for 3 days. Axial chest CT shows a small focal and central ground glass opacity (GGO) in the right upper lobe; B, female, 40 years old. Axial chest CT shows small peripheral linear opacities bilaterally. C, male, 38 years old. Axial chest CT shows a GGO in the central left lower lobe; D, male, 31 years old with fever for 1 day. Axial chest CT shows a linear opacity in the left lower lateral mid lung. Figure 3c: Examples of chest CT findings less commonly reported in COVID-19 infection (atypical) in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 36 years old with cough for 3 days. Axial chest CT shows a small focal and central ground glass opacity (GGO) in the right upper lobe; B, female, 40 years old. Axial chest CT shows small peripheral linear opacities bilaterally. C, male, 38 years old. Axial chest CT shows a GGO in the central left lower lobe; D, male, 31 years old with fever for 1 day. Axial chest CT shows a linear opacity in the left lower lateral mid lung. Figure 3d: Examples of chest CT findings less commonly reported in COVID-19 infection (atypical) in patients with epidemiological and clinical presentation suspicious for COVID-19 infection. A, male, 36 years old with cough for 3 days. Axial chest CT shows a small focal and central ground glass opacity (GGO) in the right upper lobe; B, female, 40 years old. Axial chest CT shows small peripheral linear opacities bilaterally. C, male, 38 years old. Axial chest CT shows a GGO in the central left lower lobe; D, male, 31 years old with fever for 1 day. Axial chest CT shows a linear opacity in the left lower lateral mid lung. Discussion In our series, the sensitivity of chest CT was greater than that of RT-PCR (98% vs 71%, respectively, p<.001). The reasons for the low efficiency of viral nucleic acid detection may include: 1) immature development of nucleic acid detection technology; 2) variation in detection rate from different manufacturers; 3) low patient viral load; or 4) improper clinical sampling. The reasons for the relatively lower RT-PCR detection rate in our sample compared to a prior report are unknown (3). Our results support the use of chest CT for screening for COVD-19 for patients with clinical and epidemiologic features compatible with COVID-19 infection particularly when RT-PCR testing is negative.
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            CRISPR-Cas12–based detection of SARS-CoV-2

            An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.
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              Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

              CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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                Author and article information

                Contributors
                Journal
                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                2234-943X
                07 August 2020
                2020
                07 August 2020
                : 10
                : 1387
                Affiliations
                [1] 1Department of Medicine, Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center , New York, NY, United States
                [2] 2Weill Cornell Medicine, Cornell University , New York, NY, United States
                Author notes

                Edited by: Israel Gomy, Dana–Farber Cancer Institute, United States

                Reviewed by: Nan Wu, Peking Union Medical College Hospital (CAMS), China; Tanja Kunej, University of Ljubljana, Slovenia; Martin Roffe, A.C. Camargo Cancer Center, Brazil

                *Correspondence: Charles M. Rudin rudinc@ 123456mskcc.org

                This article was submitted to Cancer Genetics, a section of the journal Frontiers in Oncology

                †Lead Contact

                Article
                10.3389/fonc.2020.01387
                7427626
                32850447
                e036d3ba-5d13-4573-827a-66ce6b30bbb5
                Copyright © 2020 Uddin, Rudin and Sen.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 09 March 2020
                : 30 June 2020
                Page count
                Figures: 4, Tables: 2, Equations: 0, References: 119, Pages: 17, Words: 11545
                Categories
                Oncology
                Review

                Oncology & Radiotherapy
                gene therapy,crispr/cas9,homology-directed repair (hdr),non-homologous end joining (nhej),clinical trial,ethics

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