Browsing by Author "Deshmukh, Harshavardhan"
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Item A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing(Elsevier, 2019) Li, Ang; Lee, Ciaran M.; Hurley, Ayrea E.; Jarrett, Kelsey E.; De Giorgi, Marco; Lu, Weiqi; Balderrama, Karol S.; Doerfler, Alexandria M.; Deshmukh, Harshavardhan; Ray, Anirban; Bao, Gang; Lagor, William R.Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.Item Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing(AAAS, 2022) Park, So Hyun; Cao, Mingming; Pan, Yidan; Davis, Timothy H.; Saxena, Lavanya; Deshmukh, Harshavardhan; Fu, Yilei; Treangen, Todd; Sheehan, Vivien A.; Bao, GangMost genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.Item Visualization of RNA virus infection in a marine protist with a universal biomarker(Springer Nature, 2023) Coy, Samantha R.; Utama, Budi; Spurlin, James W.; Kim, Julia G.; Deshmukh, Harshavardhan; Lwigale, Peter; Nagasaki, Keizo; Correa, Adrienne M. S.Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique—dsRNA-Immunofluorescence (dsRIF)—that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.