Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al 2 O 3 substrate. The adhesion is mediated by Ca 2+ in the ambient buffer. Subsequent depletion of Ca 2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al 2 O 3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation. Video Link The video component of this article can be found at https://www.jove.com/video/58923/ 4. In this method, an ER model is fabricated with a bottom-up approach using only a few elements, in which the molecules of interest can be integrated to add complexity. The network represents ER structure and dynamics. Furthermore, reversible transformation between the planar membrane and the tubes, vesicle formation from the tubes, tube fusion, sliding and retraction can all be observed. In addition to serving as a bottom-up model for the incompletely understood cellular ER, the lipid route to nanotube networks described in this protocol can be applicable for researchers studying self-assembly, nanofluidics, single-molecule and colloid

A novel cellular automaton (CA) for simulating biological membrane rupture is proposed. Constructed via simple rules governing deformation, tension, and fracture, the CA incorporates ideas from standard percolation models and bond-based fracture methods. The model is demonstrated by comparing simulations with experimental results of a double bilayer lipid membrane expanding on a solid substrate. Results indicate that the CA can capture non-trivial rupture morphologies such as floral patterns and the saltatory dynamics of fractal avalanches observed in experiments. Moreover, the CA provides insight into the poorly understood role of inter-layer adhesion, supporting the hypothesis that the density of adhesion sites governs rupture morphology.

Cas12a CRISPR technology, unlike Cas9, allows for multiplexing guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

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