A5089289021- CRISPR and Genetic Engineering
- RNA regulation and disease
- RNA and protein synthesis mechanisms
- Advanced biosensing and bioanalysis techniques
- RNA Interference and Gene Delivery
- Cytomegalovirus and herpesvirus research
- Insect symbiosis and bacterial influences
- Virus-based gene therapy research
- Chronic Lymphocytic Leukemia Research
- Mosquito-borne diseases and control
- Genomics and Phylogenetic Studies
- Monoclonal and Polyclonal Antibodies Research
- Enzyme Structure and Function
- Plant Virus Research Studies
- Osteoarthritis Treatment and Mechanisms
- Total Knee Arthroplasty Outcomes
- RNA Research and Splicing
- Knee injuries and reconstruction techniques
- Innovation and Socioeconomic Development
- Protein Structure and Dynamics
- RNA modifications and cancer
- Receptor Mechanisms and Signaling
- Immunodeficiency and Autoimmune Disorders
Beam Therapeutics (United States)
2019-2021
Broad Institute
2017-2020
Harvard University
2016-2019
Howard Hughes Medical Institute
2016-2019
Boston Children's Hospital
2018
Dana-Farber Cancer Institute
2018
Cold Spring Harbor Laboratory
2018
European Bioinformatics Institute
2015
Wellcome Trust
2015
Significance The therapeutic potential of protein-based genome editing is dependent on the delivery proteins to appropriate intracellular targets. Here we report that combining bioreducible lipid nanoparticles and negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives self-assembly for potent protein editing. design lipids facilitates degradation inside cells in response reductive environment, enhancing endosome escape protein. In addition, modulation...
Abstract We recently developed base editing, a genome-editing approach that enables the programmable conversion of one pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application editing is limited by off-target activity reliance intracellular delivery. Here we describe two advances address these limitations. First, greatly reduce installing mutations our third-generation editor (BE3) to...
Adenine base editors (ABEs) enable precise and efficient conversion of target A•T pairs to G•C in genomic DNA with a minimum by-products. While ABEs have been reported exhibit minimal off-target editing, editing cellular RNA by has not examined depth. Here, we demonstrate that current ABE generates low but detectable levels widespread adenosine-to-inosine RNAs. Using structure-guided principles design mutations both deaminase domains, developed new variants retain their ability edit...
Abstract Programmable nucleases can introduce precise changes to genomic DNA through homology-directed repair (HDR). Unfortunately, HDR is largely restricted mitotic cells, and typically accompanied by an excess of stochastic insertions deletions (indels). Here we present in vivo base editing strategy that addresses these limitations. We use nuclease-free install a S33F mutation β-catenin blocks phosphorylation, impedes degradation, upregulates Wnt signaling. In vitro, installs the with...
Abstract Cytosine base editors (CBEs) enable efficient, programmable reversion of T•A to C•G point mutations in the human genome. Recently, cytosine with rAPOBEC1 were reported induce unguided deamination genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B R54Q], or PpAPOBEC1 H122A, R33A]) that display comparable on-target editing frequencies, whilst eliciting a 12- 69-fold reduction C-to-U edits transcriptome, up...
Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations PACE contains 20 non-silent mutations, IL-23 at the peptide bond, and when pre-mixed with primary cultures murine splenocytes inhibits IL-23-mediated immune signaling. We characterize substrate specificity this evolved enzyme, revealing shifted broadened...
In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. presence homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency a low ratio HDR products:indels. Here, we develop hRad51 mutants fused Cas9(D10A) nickase (RDN) mediate while minimizing indels. We use RDN...
Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment transition mutations without double-strand DNA break intermediates. The breadth potential base editing targets is frequently limited by the requirement suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures TadA to design set inlaid (IBEs), in which domains internal Cas9. Several these IBEs exhibit shifted windows greater efficiency, enabling outside...
The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show transcript-bound increases the binding of NMD UPF1 to while in, or associated with, nucleus, bypassing UPF2 recruitment and promoting NMD. when positioned downstream PTC, which resembles mode action exon junction complex (EJC) factors. Moreover, and/or EJC deposition increase effect on Lastly, enhances PTC-containing...
Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric of known structure have even stoichiometry, a significant minority uneven stoichiometry--that is, differing numbers each subunit type. To adopt this sequence-identical subunits must be asymmetric respect to other, forming different interactions within the complex. Here we first investigate occurrence demonstrating that it is common in vitro and likely vivo. Next, elucidate structural determinants...
The foundational adenine base editors ( e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants create ABE8s. At NGG PAM sites, ABE8s result ∼1.5x higher protospacer positions A5-A7 and ∼3.2x A3-A4 A8-A10 compared with ABE7.10. Non-NGG have ∼4.2-fold overall on-target efficiency than In CD34+ cells, ABE8 recreate natural allele...
Abstract Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring efficiency evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification that incorporates biologically-informed scalable alignment approaches. Here, we present CRISPResso2 fill this gap illustrate its functionality by experimentally analyzing the properties six genome agents.
Abstract/introductory paragraph Cytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T•A to C•G point mutations in the human genome without induction DNA double strand breaks 1, 2 . Recently, foundational cytosine editor (CBE) ‘BE3’, containing rAPOBEC1, was reported induce unguided, genomic 3, 4 cellular RNA 5 deamination when expressed living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput assay...