Gene editing through microfluidic delivery
Gene editing can be performed by introducing gene-editing components into a cell by mechanical cell disruption. Related apparatus, systems, techniques, and articles are also described.
1. A microfluidic system for delivering a complex comprising a protein and a nucleic acid (protein-nucleic acid complex) into a cell, comprising: (a) at least one microfluidic channel and (b) a protein-nucleic acid complex, wherein the at least one microfluidic channel comprises a cell-deforming constriction having a diameter that is 20-99% of the diameter of the cell and allows the delivery of the protein-nucleic acid complex in an intact form.
2. The microfluidic system of claim 1 , wherein:
the microfluidic system further comprises the cell, wherein the cell is provided in a suspension; and
the microfluidic system is configured to pass the suspension comprising the cell through the microfluidic channel that includes the cell-deforming constriction such that a pressure is applied to the cell causing perturbations of the cell large enough for the protein-nucleic acid complex to pass through.
3. The microfluidic system of claim 2 , wherein the system is configured such that the cell is contacted with the protein-nucleic acid complex (a) before the cell passes through the constriction; or (b) before and during the cell passes through the constriction.
4. The microfluidic system of claim 2 , wherein the protein-nucleic acid complex comprises gene editing components.
5. The microfluidic system of claim 2 , wherein the protein comprises a ribonucleoprotein (RNP).
6. The microfluidic system of claim 2 , wherein
(a) the protein is a Cas protein or a Cpf1 protein; and
(b) the nucleic acid is a single guide RNA (sgRNA) or a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).
7. The microfluidic system of claim 6 , wherein the protein-nucleic acid complex is a RNP comprising a Cas protein or a Cpf1 protein and a sgRNA, wherein the Cas protein or the Cpf1 protein and the sgRNA were complexed using about a 0.5, 2.0, 2.5, or 3.0 molar excess of the Cas protein or Cpf1 protein.
8. The microfluidic system of claim 6 , wherein the Cas protein comprises a Cas9 protein.
9. The microfluidic system of claim 5 , wherein the protein-nucleic acid complex comprises a first RNP and a second RNP.
10. The microfluidic system of claim 9 , wherein the first RNP and the second RNP are both nickases.
11. The microfluidic system of claim 10 , wherein the first RNP nicks a target sequence different from the target sequence of the second RNP.
12. The microfluidic system of claim 2 , wherein the protein comprises a TALEN protein, Zinc finger nuclease, mega nuclease, or Cre recombinase.
13. The microfluidic system of claim 2 , wherein the nucleic acid comprises an mRNA encoding a TALEN protein, a Zinc finger nuclease, a mega nuclease, or a Cre recombinase.
14. The microfluidic system of claim 2 , wherein the protein-nucleic acid complex comprises
(a) a nucleic acid molecule that is complexed with a protein via electrostatic attraction;
(b) a nucleic acid molecule wrapped around a protein;
(c) DNA and a histone;
(d) a ribonucleoprotein (RNP);
(e) a ribosome, an enzyme telomerase, a vault ribonucleoprotein, RNase P, hnRNP, or a small nuclear RNP (snRNP); or
(f) a chromosome comprising a protein.
15. The microfluidic system of claim 2 , wherein the suspension further comprises a donor DNA.
16. The microfluidic system of claim 15 , wherein the suspension comprises the donor DNA before, during, and/or after the cell passes through the constriction.
17. The microfluidic system of claim 2 , wherein the cell comprises a mammalian cell.
18. The microfluidic system of claim 2 , wherein the cell comprises a human cell.
19. The microfluidic system of claim 2 , wherein a diameter of the constriction is about 60% of the diameter of the cell.
20. The microfluidic system of claim 2 , wherein the microfluidic channel is one of a plurality of parallel microfluidic channels in the microfluidic system.
21. The microfluidic system of claim 20 , wherein the plurality of parallel microfluidic channels comprises at least about 2, 5, 10, 20, 25, 30, 40, 45, 50, 75, 100, 500, 1,000, or 2-1,000 microfluidic channels.
22. The microfluidic system of claim 2 , wherein the cell is a plurality of cells, and each cell is passed through one of a plurality of parallel microfluidic channels, and wherein each microfluidic channel of the plurality of parallel microfluidic channels includes a cell-deforming constriction.
23. The microfluidic system of claim 2 , wherein
(a) the diameter of the constriction is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2-10 μm, or 10-20 μm;
(b) the length of the constriction is about 10, 15, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100, 10-40, 10-50, 10-60, or 10-100 μm;
(c) a pressure of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 10-100 psi is used to pass the suspension through the microfluidic channel;
(d) the cell passes through the microfluidic channel at a speed of about 300, 400, 500, 600, 700, 800, 900, 100-300, 200-700, 250-400, 100-1000 mm/s, 1-1000 mm/s, 1 m/s, 2 m/s, 3 m/s, 4 m/s, 5 m/s, 6 m/s, 7 m/s, 8 m/s, 9 m/s, 10 m/s, 0.01-5 m/s, 5-10 m/s, or 0.01-10 m/s;
(e) the microfluidic channel comprises multiple cell-deforming constrictions in series;
(f) the microfluidic channel comprises a single cell-deforming constriction;
(g) the perturbations of the cell membrane include a maximum diameter of about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 nm; and/or
(h) perturbations of the cell membrane having a maximum diameter of about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 nm persist on the cell membrane for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10 minutes.