Biology subjects

Katrekar, D.

Publications and source records attributed to Katrekar, D..

4 recordsLinked to original sources

bioRxiv2025

Megabase-scale human genome rearrangement with programmable bridge recombinases

Bridge recombinases are a class of naturally occurring RNA-guided DNA recombinases. We previously demonstrated they can programmably insert, excise, and invert DNA in vitro and in bacteria. Here, we report the discovery and engineering of IS622, a simple two-component system capable of universal DNA rearrangements of the human genome. We define strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the IS622 bridge RNA and deep mutational scanning of its recombinase, we achieve up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrate intra-chromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Finally, we provide proof-of-concept for excision of a gene regulatory region or expanded repeats relevant for the treatment of genetic diseases.

bioengineering↗

bioRxiv2021

Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Akin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

bioengineering↗

bioRxiv2020

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5 guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5-GA-3 motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

synthetic biology↗

bioRxiv2017

In vivo RNA targeting of point mutations via suppressor tRNAs and adenosine deaminases

Point mutations underlie many genetic diseases. In this regard, while programmable DNA nucleases have been used to repair mutations, their use for gene therapy poses multiple challenges: one, efficiency of homologous recombination is typically low in cells; two, an active nuclease presents a risk of introducing permanent off-target mutations; and three, prevalent programmable nucleases typically comprise elements of non-human origin raising the potential of in vivo immunogenicity. In light of these, approaches to instead directly target RNA, and use of molecular machinery native to the host would be highly desirable. Towards this, we engineered and optimized two complementary approaches, referred together hereon as tRiAD, based on the use of tRNAs in codon suppression and adenosine deaminases in RNA editing. Specifically, by delivering modified endogenous tRNAs and/or the RNA editing enzyme ADAR2 and an associated guiding RNA (adRNA) via adeno-associated viruses, we enabled premature stop codon read-through and correction in the mdx mouse model of muscular dystrophy that harbors a nonsense mutation in the dystrophin gene. We further demonstrated inducible restoration of dystrophin expression by pyrolysyl-tRNA mediated incorporation of unnatural amino acids (UAAs) at the stop codon. Additionally, we also engineered ADAR2 mediated correction of a point mutation in liver RNA of the spfash mouse model of ornithine transcarbamylase (OTC) deficiency. Taken together, our results establish the use of suppressor tRNAs and ADAR2 for in vivo RNA targeting, and this integrated tRiAD approach is robust, genomically scarless, and potentially non-immunogenic as it utilizes effector RNAs and human proteins.

synthetic biology↗