Methods, systems and devices for selection and generation of genome edited clones
View Patent ↗Methods are described herein for isolating clonal populations of cells having a defined genetic modification. The methods are performed, at least in part, in a microfluidic device comprising one or more sequestration pens. The methods include the steps of: maintaining individual cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the individual cells into respective clonal populations of cells; and detecting, in one or more cells of each clonal population, the presence of a first nucleic acid sequence that is indicative of the presence of an on-target genome edit in the clonal population of cells. Also described are methods of performing genome editing within a microfluidic device, and compositions comprising one or more clonal populations of cells generated according to the methods disclosed herein.
1. A method of generating a clonal population of genetically modified cells in a microfluidic device comprising a sequestration pen, the method comprising:
maintaining a first cell in the sequestration pen of the microfluidic device, wherein the first cell has undergone a genome editing process;
expanding the first cell into a clonal population of cells;
detecting, in a first subset of the clonal population of cells, the presence of a first nucleic acid sequence by extracting nucleic acid from the first subset, wherein the first subset comprises one or more cells and the first nucleic acid sequence indicates the presence of an on-target genome edit in the clonal population of cells; and
preserving a second subset of the clonal population of cells.
2. The method of claim 1 , wherein the first cell is a mammalian cell.
3. The method of claim 1 , wherein the first cell is a stem cell, a progenitor cell, or an immunological cell.
4. The method of claim 1 , further comprising:
contacting the first cell with a genome editing biomolecule; and
introducing the first cell into the microfluidic device.
5. The method of claim 4 , further comprising:
contacting the first cell with a donor template nucleic acid molecule.
6. The method of claim 5 , wherein the donor template nucleic acid molecule comprises all or part of the first nucleic acid sequence.
7. The method of claim 4 , wherein the step of contacting the first cell with the genome editing biomolecule is performed prior to the step of introducing the first cell into the microfluidic device.
8. The method of claim 1 , wherein the microfluidic device comprises a substrate having a DEP configuration, and
wherein the method further comprises positioning the first cell in the sequestration pen using dielectrophoretic (DEP) force.
9. The method claim 1 , wherein
the extracted nucleic acid comprises genomic DNA.
10. The method of claim 9 , further comprising:
moving the first subset of cells out of the sequestration pen; and
exporting the first subset of cells from the microfluidic device, wherein the nucleic acid is extracted outside of the microfluidic device.
11. The method of claim 9 , further comprising:
moving the first subset of cells out of the sequestration pen to a separate region within the microfluidic device, wherein the nucleic acid is extracted in the separate region.
12. The method of claim 9 , further comprising amplifying the extracted nucleic acid.
13. The method of claim 1 , wherein the on-target genome edit comprises a deletion of endogenous deoxyribonucleic acid (DNA) at a target site in the genome and/or an insertion of exogenous deoxyribonucleic acid (DNA) at the target site in the genome.
14. The method of claim 1 , further comprising:
detecting, in one of more cells of the clonal population, the presence of a second nucleic acid sequence, wherein the combination of the first nucleic acid sequence and the second nucleic acid sequence indicates the presence of the on-target genome edit in the clonal population of cells.
15. The method of claim 1 , further comprising:
detecting, in one of more cells of the clonal population of cells, the presence of an additional nucleic acid sequence, wherein the additional nucleic acid sequence indicates the presence of an off-target genome edit in the clonal population of cells.
16. The method of claim 1 , wherein expanding the first cell into a clonal population of cells further comprises monitoring one or more characteristics of the cells of the clonal population for a period of time.
17. The method of claim 16 , wherein the monitoring comprises assessing the production of a protein of interest, the presence of one or more cell surface markers, and/or reaction with a specific antibody.
18. A method of generating a clonal population of genetically modified cells in a microfluidic device comprising a sequestration pen, the method comprising:
maintaining a first cell in the sequestration pen of the microfluidic device, wherein the first cell has undergone a genome editing process;
expanding the first cell into a clonal population of cells; and
detecting, in one or more cells of the clonal population, the presence of a first nucleic acid sequence,
wherein at least one inner surface of the sequestration pen is a conditioned surface comprising covalently-linked molecules, each having a linking group (LG) covalently bound to the at least one inner surface and a moiety covalently bound to the linking group (LG) via a linker (L) having a linear backbone of 10 to 20 carbon atoms,
wherein the moieties of the covalently-linked molecules include amino acid-containing polymers suitable for maintenance and/or expansion of the genome-edited cell, and
wherein the first nucleic acid sequence indicates the presence of an on-target genome edit in the clonal population of cells.
19. The method of claim 1 , wherein the microfluidic device comprises a plurality of sequestration pens, and wherein the method is performed on a plurality of cells to thereby generate a plurality of clonal populations of genetically modified cells.
20. The method of claim 1 , wherein the microfluidic device comprises an enclosure comprising:
a flow region for containing a flow of a first fluidic medium; and
the sequestration pen, wherein the sequestration pen comprises:
an isolation region for containing a second fluidic medium, the isolation region having a single opening; and
a connection region fluidically connecting the isolation region to the flow region;
wherein the isolation region of the sequestration pen is an unswept region of the microfluidic device.
21. The method of claim 20 , wherein the enclosure comprises a microfluidic channel, and at least a portion of the flow region is comprised by the microfluidic channel; and further wherein the connection region comprises a proximal opening into the microfluidic channel having a width W con ranging from about 20 microns to about 100 microns and a distal opening into said isolation region, and wherein a length L con of said connection region from the proximal opening to the distal opening is as least 1.0 times a width W con of the proximal opening of the connection region.
22. The method of claim 21 , wherein the length L con of the connection region from the proximal opening to the distal opening is at least 1.5 times the width W con of the proximal opening of the connection region.
23. The method of claim 21 , wherein the length L con of the connection region from the proximal opening to the distal opening is at least 2.0 times the width W con of the proximal opening of the connection region.
24. The method of claim 21 , wherein the width W con of the proximal opening of the connection region ranges from about 20 microns to about 60 microns.
25. The method of claim 21 , wherein (i) the length L con of the connection region from the proximal opening to the distal opening is between about 20 microns and about 500 microns; (ii) a width of the microfluidic channel at the proximal opening of the connection region is between about 50 microns and about 500 microns; and/or (iii) the proximal opening of the connection region is parallel to a direction of the flow of the first medium in the flow region.
26. The method of claim 21 , wherein a height of the microfluidic channel at the proximal opening of the connection region is between 20 microns and 100 microns.
27. The method of claim 1 , wherein the first cell is a human cell.
28. The method of claim 18 , wherein the amino acid-containing polymers are extracellular matrix proteins.
29. The method of claim 18 , wherein the moieties of the covalently-linked molecules include a mixture of polymers, the mixture further comprising polymers having alkylene oxide moieties.
30. The method of claim 18 , wherein the moieties of the covalently linked molecules are covalently bound to the linker (L) via a coupling group (CG).