7. Count the cells.
8. Pellet the appropriate number of cells by centrifugation at
400 Â g for 5 min at 4
C.
9. Aspirate the PBS carefully and resuspend the cell pellet in
electroporation buffer at 5000 cells per μL.
10. For single-color labeling, add 1 μL of the 10 μM MTSatargeting or MTSb-targeting MB solution for every 10 μL of
cells to achieve a final MB concentration of 1 μM. For dualcolor labeling, add 1 μL of the MTSa-targeting MB solution
and 1 μL of the MTSb-targeting MB solution for every 10 μL
of cells to achieve a final MB concentration of 2 μM.
11. Pipette to mix the cells with MBs.
12. Add 4 mL electroporation buffer into the electroporation tube.
13. Microporate 10 μL of the cell suspension in an electroporation
chamber at 1150 V with a 20 ms pulse width and 2 pulses total
(see Note 19).
14. Gently transfer the microporated cells from the tip to a microcentrifuge tube prefilled with 1.5 mL of fresh cell culture
medium (see Note 18).
15. Pellet the cells by centrifugation at 400 Â g for 5 min at 4
C.
16. Aspirate the medium to avoid disturbing the cell pellet (see
Note 20). Resuspend the pellet gently with 1.5 mL medium.
17. Repeat steps 15 and 16 two more times.
18. After the last wash, resuspend the cells in 250 μL of cell culture
medium.
19. Seed the cells into a well of an 8-well chambered cover glass
coated with fibronectin (see Note 21).
20. Incubate the cells in a cell incubator (37
C, 5% (v/v) CO 2 , and
90% relative humidity). Cells can be imaged after sufficient
time is given to allow cells to attach and spread on the coverslip
(approximately 8 h).
3.3 Validation
of CRISPR/MB Signals
by DNA FISH
It is important to validate the specificity of CRISPR/MB signal to
test the labeling accuracy and to exclude nonspecific signals. One
straightforward validation method is to use DNA FISH to label the
same locus and determine if the resulting signal colocalizes with the
CRISPR/MB signal. Note that because of the helicase activity of
dCas9 to unwind the target DNA duplex, DNA FISH can be
performed without the high-temperature heating employed in
conventional DNA FISH protocols. The detailed protocol is
shown below:
364
Xiaotian Wu et al.
8. Pellet the appropriate number of cells by centrifugation at
400 Â g for 5 min at 4
C.
9. Aspirate the PBS carefully and resuspend the cell pellet in
electroporation buffer at 5000 cells per μL.
10. For single-color labeling, add 1 μL of the 10 μM MTSatargeting or MTSb-targeting MB solution for every 10 μL of
cells to achieve a final MB concentration of 1 μM. For dualcolor labeling, add 1 μL of the MTSa-targeting MB solution
and 1 μL of the MTSb-targeting MB solution for every 10 μL
of cells to achieve a final MB concentration of 2 μM.
11. Pipette to mix the cells with MBs.
12. Add 4 mL electroporation buffer into the electroporation tube.
13. Microporate 10 μL of the cell suspension in an electroporation
chamber at 1150 V with a 20 ms pulse width and 2 pulses total
(see Note 19).
14. Gently transfer the microporated cells from the tip to a microcentrifuge tube prefilled with 1.5 mL of fresh cell culture
medium (see Note 18).
15. Pellet the cells by centrifugation at 400 Â g for 5 min at 4
C.
16. Aspirate the medium to avoid disturbing the cell pellet (see
Note 20). Resuspend the pellet gently with 1.5 mL medium.
17. Repeat steps 15 and 16 two more times.
18. After the last wash, resuspend the cells in 250 μL of cell culture
medium.
19. Seed the cells into a well of an 8-well chambered cover glass
coated with fibronectin (see Note 21).
20. Incubate the cells in a cell incubator (37
C, 5% (v/v) CO 2 , and
90% relative humidity). Cells can be imaged after sufficient
time is given to allow cells to attach and spread on the coverslip
(approximately 8 h).
3.3 Validation
of CRISPR/MB Signals
by DNA FISH
It is important to validate the specificity of CRISPR/MB signal to
test the labeling accuracy and to exclude nonspecific signals. One
straightforward validation method is to use DNA FISH to label the
same locus and determine if the resulting signal colocalizes with the
CRISPR/MB signal. Note that because of the helicase activity of
dCas9 to unwind the target DNA duplex, DNA FISH can be
performed without the high-temperature heating employed in
conventional DNA FISH protocols. The detailed protocol is
shown below:
364
Xiaotian Wu et al.