RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9–mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.

Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

In the development of the mammalian telencephalon, the genesis of neurons destined for the various layers of the cerebral cortex is preceded by the generation of a population of cells that comes to reside in the subplate and marginal zones (see ref. 2 for nomenclature). In the cat, these cells are present in large numbers during development, when their location is correlated with the arrival and accumulation of ingrowing axonal systems and with synapses. However, as the brain matures, the cells disappear and the white matter and layer 1 of the adult emerge. Their disappearance occurs in concert with the invasion of the cortical plate by the axonal systems and with the elimination of the synapses from the subplate. Here we report that the subplate cells have properties typical of mature neurons. They have the ultrastructural appearance of neurons and receive synaptic contacts. They also have long projections and are immunoreactive for MAP2 (microtubule associated protein 2). Further, subpopulations are immunoreactive for one of several neuropeptides. These observations suggest that during the fetal and early postnatal development of the mammalian telencephalon the subplate cells function as neurons in synaptic circuitry that disappears by adulthood.

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