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rabindra.tharu.np@gmail.com
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Development of compact transcriptional effectors using high-throughput measurements in diverse contexts
Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector…
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rabindra.tharu.np@gmail.com
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Comprehensive genome analysis and variant detection at scale using DRAGEN
Research and medical genomics require comprehensive, scalable methods for the discovery of novel disease targets, evolutionary drivers and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size or location. Here we present DRAGEN, which uses multigenome mapping with pangenome references, hardware acceleration and machine learning-based…
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rabindra.tharu.np@gmail.com
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Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection
CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants…
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rabindra.tharu.np@gmail.com
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Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView
Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro…
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rabindra.tharu.np@gmail.com
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Rapid generation of long, chemically modified pegRNAs for prime editing
The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint…
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rabindra.tharu.np@gmail.com
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Continuous multiplexed phage genome editing using recombitrons
Bacteriophage genome editing can enhance the efficacy of phages to eliminate pathogenic bacteria in patients and in the environment. However, current methods for editing phage genomes require laborious screening, counterselection or in vitro construction of modified genomes. Here, we present a scalable approach that uses modified bacterial retrons called recombitrons to generate recombineering donor DNA…
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rabindra.tharu.np@gmail.com
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A CAR enhancer increases the activity and persistence of CAR T cells
Although chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical outcomes, durable remissions remain limited. To extend the efficacy of CAR T cells, we develop a CAR enhancer (CAR-E), comprising a CAR T cell antigen fused to an immunomodulatory molecule. Here we demonstrate this strategy using B cell maturation antigen (BCMA) CAR T…
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rabindra.tharu.np@gmail.com
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Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases
Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions.…
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rabindra.tharu.np@gmail.com
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Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency
The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against…
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rabindra.tharu.np@gmail.com
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Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing
The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA…
