Biology subjects

Richey, J.

Publications and source records attributed to Richey, J..

3 recordsLinked to original sources

bioRxiv2024

Efficient creation and characterization of multi-kilobase knockins by CRISPR/Cas9 and multiple rAAVs

Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex (RNP) enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.6 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared to other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of the KI allele, genotypes non-KI alleles and more importantly, uniquely identifies donor concatenation in the KI allele, and localizes random integration of the full or partial donor. Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/625457v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1619b77org.highwire.dtl.DTLVardef@125b0b2org.highwire.dtl.DTLVardef@173b12dorg.highwire.dtl.DTLVardef@7519b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis

DNA methylation has been utilized for target gene silencing in plants, however its not well-understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain of function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression either through the establishment of DNA methylation, or via DNA methylation-independent processes including histone H3K27me3 deposition, H3K4me3 demethylation, H3K9, H3K14, H3K27, and H4K16 deacetylation, inhibition of RNA Polymerase II transcription elongation or Ser-5 dephosphorylation. The silencing fusion proteins also silenced many other genes with different efficacy, and a machine learning model could accurately predict the efficacy of each silencer based on various chromatin features of the target loci. These results provide a more comprehensive understanding of epigenetic regulatory pathways and provide an armament of tools for targeted manipulation of gene expression.

plant biology↗

bioRxiv2020

Mapping social reward and punishment processing in the human brain: A voxel-based meta-analysis of neuroimaging findings using the Social Incentive Delay task

Social incentives (rewards or punishments) motivate human learning and behaviour, and alterations in the brain circuits involved in the processing social incentives have been linked with several neuropsychiatric disorders. However, questions still remain about the exact neural substrates implicated in social incentive processing. Here, we conducted four Anisotropic Effect Size Signed Differential Mapping voxel-based meta-analyses of fMRI studies investigating the neural correlates of the anticipation and receipt of social rewards and punishments using the Social Incentive Delay task. We map the regions involved in each of these four processes in the human brain, identify decreases in the BOLD signal during the anticipation of both social reward and punishment avoidance that were missed in individual studies due to a lack of power, and characterise the effect size and direction of changes in the BOLD signal for each brain area. Our results provide a better understanding of the brain circuitry involved in social incentive processing and can inform hypotheses about potentially disrupted brain areas linked with dysfunctional social incentive processing during disease. HighlightsO_LIVoxel-based meta-analysis of the neural underpinnings of social incentive processing C_LIO_LIWe map the brain regions involved in the processing of social incentives in humans C_LIO_LIWe identify new regions missed in individual studies as a result of lack of power C_LIO_LIOur work can inform research on pathological brain processing of social incentives C_LI

neuroscience↗