Abstract: Provided is a method of generating droplets that includes aspirating a first liquid into a tube, positioning the tube over a receiving liquid, and ejecting the first liquid to generate a plurality of droplets that contact the receiving liquid and remain discrete even after contacting the receiving liquid. Whereas many other droplet generators require complex microfluidics, the present methods allow generation of droplets without the need for microfluidics. The methods can be performed with existing commercially available macro-fluidic liquid handling devices. The methods can be used for digital PCR, digital MDA, enzyme screening, single cell analysis, and other applications involving droplets.

Abstract: Nucleic acid-guided nuclease editing in mammalian cells may include passaging mammalian cells, in an automated closed cell editing instrument, into smaller aggregates when the aggregates exceed 50-300 microns in size. A library of viral particles may be delivered to the mammalian cells at a multiplicity of infection such that each mammalian cell receives one or no viral particle. The library may include viral vectors with an editing cassette including a pair of gRNA coding sequence and donor DNA. Conditions may be provided to allow a viral vector of the viral vectors to integrate into the mammalian cells. Enriching for mammalian cells may be done with an integrated viral vector. A nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion may be delivered to the enriched mammalian cells and conditions may be provided to allow editing in the mammalian cells.

Abstract: Compositions of matter, methods, modules, and automated instruments may relate to synthesizing a library including an editing cassette including a different gRNA and donor DNA pair, amplifying the editing cassette in a partition separate from other editing cassettes in the library, adding nuclease to the partition, and adding lipofectamine to the editing cassette and nuclease to form a lipofectamine/nucleic acid/nuclease complex. A microcarrier coated in extracellular matrix or a cell adhesion molecule coating may be added to the lipofectamine/nucleic acid/nuclease complex. Cell growth material, the microcarrier, and mammalian cells may be transferred to a growth module in an automated closed cell editing instrument via a liquid handling system. The mammalian cells may be allowed to seed on the microcarrier. Conditions may be provided for the mammalian cells to take-up and be edited by a payload associated with the lipofectamine/nucleic acid/nuclease complex.

Abstract: The present disclosure relates to automated multi-module instruments, compositions and methods for performing nucleic acid-guided nuclease editing; specifically, the disclosure provides nucleic acid cassettes, plasmids, vectors, and compositions comprising the same that employ homologous recombination for genome engineering by having a CRISPR nuclease cause a specific DSB while tethered to a repair nucleic acid.

Abstract: The present disclosure provides a sphere-packing lattice electroporation device configured for use as a stand-alone unit or in an automated multi-module cell processing environment and configured to decrease cell processing time and cell survival. The sphere-packing lattice utilizes lattice-forming beads that are uniform in size and that self-assemble into a crystalline-like lattice.

Abstract: This invention relates to compositions of matter, methods, modules and automated, end-to-end closed instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells. The disclosed compositions and method entail making “reagent bundles” comprising many (hundreds of thousands to millions) clonal copies of an editing cassette and delivering or co-localizing the reagent bundles with live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells. The present compositions and methods entail viral delivery of an editing cassette to live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow, preferably using a fully-automated end-to-end instrument to process the cells without human intervention to enhance cell processing uniformity and to maintain the integrity of the cell culture.

Abstract: Methods for selectively combining discrete entities including, e.g. cells, reagents, drugs, hydrogels, extracellular matrices beads, particles, biological materials, media, or a combination thereof, are provided. In certain aspects, the methods include sorting a plurality of discrete entities and trapping two or more discrete entities for a time sufficient for the two or more discrete entities to combine to form a combined discrete entity. In certain aspects, the methods include making the plurality of discrete entities. In certain aspects, the methods include detecting or analyzing the discrete entities, e.g. via optical detection. In certain aspects, the methods include manipulating or analyzing the combined discrete entity or a component therein, e.g. imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells.

Abstract: The present disclosure relates to automated multi-module instruments, compositions and methods for performing nucleic acid-guided nuclease editing; specifically, the disclosure provides nucleic acid cassettes, plasmids, vectors, and compositions comprising the same that employ homologous recombination for genome engineering by having a CRISPR nuclease cause a specific DSB while tethered to a repair nucleic acid.

Abstract: The present disclosure provides a sphere-packing lattice electroporation device configured for use as a stand-alone unit or in an automated multi-module cell processing environment and configured to decrease cell processing time and cell survival. The sphere-packing lattice utilizes lattice-forming beads that are uniform in size and that self-assemble into a crystalline-like lattice.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells. The present compositions and methods entail viral delivery of an editing cassette to live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow, preferably using a fully-automated end-to-end instrument to process the cells without human intervention to enhance cell processing uniformity and to maintain the integrity of the cell culture.

Abstract: The present disclosure relates to methods for performing arrayed nucleic acid-guided nuclease nickase fusion editing allowing for rapid genotypic/phenotypic correlation without sequencing.

Abstract: The present disclosure provides a sphere-packing lattice electroporation device configured for use as a stand-alone unit or in an automated multi-module cell processing environment and configured to decrease cell processing time and increase cell survival. The sphere-packing lattice utilizes lattice-forming beads that are uniform in size and that self-assemble into a crystalline-like lattice.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.

Abstract: This invention relates to compositions of matter, methods, modules and automated, end-to-end closed instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.

Abstract: This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells.

Abstract: The present disclosure provides a sphere-packing lattice electroporation device configured for use as a stand-alone unit or in an automated multi-module cell processing environment and configured to decrease cell processing time and cell survival.