The invention relates to a microfluidic poration device having narrow channels slightly smaller than the width of a target cell, wherein the channels are lined with a plurality of nanospikes in a row extending down the middle of the channel, i.e. in a row parallel to the sides of the channel. In one embodiment, one channel may have 2 nanospikes (or 2 nanolancets). Thus, in particular embodiments, the invention provides microfluidic poration devices capable of simultaneously squeezing cells while piercing holes in their membranes for allowing foreign molecules into cells. The holes in porated cells spontaneously close after exiting the channels, thus entrapping the foreign molecules inside of the target cells. This porated cell population has approximately a 95% viability with greater than 50% containing at least one foreign molecule.

The invention relates to a microfluidic poration device having narrow channels slightly smaller than the width of a target cell, wherein the channels are lined with a plurality of nanospikes in a row extending down the middle of the channel, i.e. in a row parallel to the sides of the channel. In one embodiment, one channel may have 2 nanospikes (or 2 nanolancets). Thus, in particular embodiments, the invention provides microfluidic poration devices capable of simultaneously squeezing cells while piercing holes in their membranes for allowing foreign molecules into cells. The holes in porated cells spontaneously close after exiting the channels, thus entrapping the foreign molecules inside of the target cells. This porated cell population has approximately a 95% viability with greater than 50% containing at least one foreign molecule.

The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

Delivering a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the payload and alcohol content greater than 5 percent concentration. The volume of the aqueous solution may be a function of exposed surface area of the population of cells, or may be a function of a number of cells in the population of cells. Related compositions, apparatus, systems, techniques, and articles are also described.

A device is provided, comprising a cell trap comprising a plurality of micro-chambers, each micro-chamber configured to hold a cell. The device can further comprise a manipulator array comprising a plurality of manipulators, each manipulator in spatial communication with a respective micro-chamber, wherein each manipulator comprises a needle, a stage, and an actuator, wherein the needle is mounted to the stage, and the actuator is operable to apply force to the stage in a direction to move the needle to penetrate a cell in the respective micro-chamber.

A device is provided, comprising a cell trap comprising a plurality of micro-chambers, each micro-chamber configured to hold a cell. The device can further comprise a manipulator array comprising a plurality of manipulators, each manipulator in spatial communication with a respective micro-chamber, wherein each manipulator comprises a needle, a stage, and an actuator, wherein the needle is mounted to the stage, and the actuator is operable to apply force to the stage in a direction to move the needle to penetrate a cell in the respective micro-chamber.

The present invention enables fabrication and mass production of a bubble-jetting chip that includes a desired number of bubble jetting portions of the same size having bubble-jetting outlets of the same size.

Mass production is enabled by fabricating a bubble-jetting chip comprising a substrate and a bubble-jetting portion formed on the substrate, the bubble-jetting portion comprising: an electrode that is formed of a conductive material; an insulating portion that is formed of an insulating photosensitive resin, is provided so as to sandwich the electrode, and includes an extended section that extends beyond the tip of the electrode; and a space that is formed between the extended section of the insulating portion and the tip of the electrode.

The present invention relates to a method of producing a non-human, mammalian oocyte carrying a modified target sequence in its genome, the method comprising the steps of introducing into a non-human, mammalian oocyte: (a) a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9 protein) or a nucleic acid molecule encoding said Cas9 protein; and (b-i) a target sequence specific CRISPR RNA (crRNA) and a trans-activating crRNA (tracr RNA) or a nucleic acid molecule encoding said RNAs; or (b-ii) a chimaeric RNA sequence comprising a target sequence specific crRNA and tracrRNA or a nucleic acid molecule encoding said RNA; wherein the Cas9 protein introduced in (a) and the RNA sequence(s) introduced in (b-i) or (b-ii) form a protein/RNA complex that specifically binds to the target sequence and introduces a single or double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the target sequence is modified by homologous recombination with a donor nucleic acid sequence further comprising the step: (c) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.

The present invention relates to a method of producing a non-human, mammalian oocyte carrying a modified target sequence in its genome, the method comprising the steps of introducing into a non-human, mammalian oocyte: (a) a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9 protein) or a nucleic acid molecule encoding said Cas9 protein; and (b-i) a target sequence specific CRISPR RNA (crRNA) and a trans-activating crRNA (tracr RNA) or a nucleic acid molecule encoding said RNAs; or (b-ii) a chimaeric RNA sequence comprising a target sequence specific crRNA and tracrRNA or a nucleic acid molecule encoding said RNA; wherein the Cas9 protein introduced in (a) and the RNA sequence(s) introduced in (b-i) or (b-ii) form a protein/RNA complex that specifically binds to the target sequence and introduces a single or double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the target sequence is modified by homologous recombination with a donor nucleic acid sequence further comprising the step: (c) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.