Akella, S. | Search bioRxiv

bioRxiv2023

Measurable fields to spikes causality and its dependence on cortical layer and area.

Distinct dynamics in different cortical layers are apparent in neuronal and local field potential (LFP) patterns, yet their associations in the context of laminar processing have been sparingly analyzed. Here, we study the laminar organization of spike-field causal flow within and across visual (V4) and frontal areas (PFC) of monkeys performing a visual task. Using an event-based quantification of LFPs and a directed information estimator, we found area and frequency specificity in the laminar organization of spike-field causal connectivity. Gamma bursts (40-80 Hz) in the superficial layers of V4 largely drove intralaminar spiking. These gamma influences also fed forward up the cortical hierarchy to modulate laminar spiking in PFC. In PFC, the direction of intralaminar information flow was from spikes [->] fields where these influences dually controlled top-down and bottom-up processing. Our results, enabled by innovative methodologies, emphasize the complexities of spike-field causal interactions amongst multiple brain areas and behavior.

neuroscience↗

bioRxiv2021

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in [~]1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures. One sentence summaryCo-targeting two genes by co-electroporation of CRISPR/Cas9 RNPs and ssODN repair templates allows concomitant genome editing to create a selectable marker gene and to introduce precise modifications in another gene of interest.

genetics↗