Biology subjects

Barrera, L. A.

Publications and source records attributed to Barrera, L. A..

5 recordsLinked to original sources

bioRxiv2023

High-efficiency base editing for Stargardt disease in mice, non-human primates, and human retina tissue

Stargardt disease is a currently untreatable, inherited neurodegenerative disease that leads to macular degeneration and blindness due to loss-of-function mutations in the ABCA4 gene. We have designed a dual adeno-associated viral vector split-intein adenine base-editing strategy to correct the most common mutation in ABCA4 (c.5882G>A, p.G1961E). We optimized ABCA4 base editing in human models, including retinal organoids, iPSC-derived retinal pigment epithelial (RPE) cells, as well as adult human retinal- and RPE/choroid explants in vitro. The resulting gene therapy vectors achieved high levels of gene correction in mutation-carrying mice and in non-human primates, with an average editing of 37% of photoreceptors and 73% of RPE cells in vivo. The high editing rates in primates make way for precise and efficient gene editing in other neurodegenerative ocular diseases.

neuroscience↗

bioRxiv2020

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

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 to create ABE8s. At NGG PAM sites, ABE8s result in [~]1.5x higher editing at protospacer positions A5-A7 and [~]3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a [~]4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the {gamma}-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

genomics↗

bioRxiv2020

Next-generation cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

Abstract/introductory paragraphCytosine 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 of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) BE3, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

genomics↗

bioRxiv2018

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

We report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporated randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

bioengineering↗

bioRxiv2017

The experimental design and data interpretation in “Unexpected mutations after CRISPR–Cas9 editing in vivo” by Schaefer et al. are insufficient to support the conclusions drawn by the authors

To the Editor To the Editor Conflict of Interest Statement References References The recent correspondence to the Editor of Nature Methods by Schaefer et al.1 has garnered significant attention since its publication as a result of its strong conclusions contradicting numerous publications in the field using similar analytical approaches and methods2-4. The authors suggest that the CRISPR-Cas9 system is highly mutagenic in genomic regions not expected to be targeted by the gRNA. We believe that the conclusions drawn from this study are unsubstantiated by the disclosed experiments as they were designed and carried out. Further, it is impossible to ascribe the observed differences in the subject mice to the effects of CRISPR per se. The genetic differences seen in ...

genetics↗