separate promoters. Amplify the U6-sgRNA cassette from
pU6-SL2-sgTelo-MTSa (see Subheading 2.3, Primer pair 2)
and the CMV-EGFP cassette from pEGFP-C1 (see Subheading
2.3, Primer pair 3). Use Gibson Assembly to simultaneously
clone the two PCR products into AseI-digested pdCas9-C1 to
create
SL2-sgTelo-MTSa/EGFP/pdCas9-C1.
Generate
SL2-sgTelo-MTSa/BFP/pdCas9-C1 similarly with the
CMV-BFP cassette PCR amplified from pmTagBFP2-C1 plasmid (see Subheading 2.3, Primer pair 4).
3. For centromere labeling, SL2-sgSat-MTSb/BFP/pdCas9-C1
should be similarly generated as SL2-sgTelo-MTSa/BFP/
pdCas9-C1. The U6-sgRNA cassette is PCR amplified from
pU6-SL2-sgSat-MTSb (see Subheading 2.3, Primer pair 5).
3.2 Cellular Delivery
of MBs
Deliver MBs into transfected HEK293 cells by microporation. The
detailed protocol is as follows:
Day 0:
Seed the proper number of HEK293 cells into a 6-well plate
with cell culture medium such that cells will reach 50–70% confluency on day 1 for transfection.
Day 1:
1. Add 200 μL fibronectin into each well of the 8-well chambered
cover glass and incubate at 37
C overnight (see Note 15).
2. Transfect the cells seeded in the 6-well plate with 2 μg of
SL2-sgTelo-MTSa/EGFP/pdCas9-C1 for telomere labeling
or SL2-sgSat-MTSb/BFP/pdCas9-C1 for centromere labeling. Transfect the cells with SL2-sgTelo-MTSa/BFP/
pdCas9-C1 and SL2-sgSat-MTSb/BFP/pdCas9-C1 at a 1:1
ratio (1 μg each) for dual labeling (see Note 16).
Day 2:
1. Aspirate the cell culture medium and incubate the cells with
1 mL pre-warmed PBS at room temperature (RT) for 2 min.
2. Aspirate the PBS and add 0.5 mL of trypsin. Incubate for 1 min
at RT.
3. Aspirate the trypsin (leaving a trace amount). Incubate at 37
C
until all cells are detached from the plate surface.
4. Neutralize the remaining trypsin with ~1 mL of cell culture
medium and gently pipette to resuspend the cells (see
Note 17).
5. Transfer the cell suspension to a 1.5 mL microcentrifuge tube
and pellet the cells by centrifugation at 400 Â g for 5 min at
4
C (see Note 18).
6. Aspirate the medium and gently resuspend the cell pellet with
1 mL of PBS.
Visualizing Genomic Loci by CRISPR/MB
363
pU6-SL2-sgTelo-MTSa (see Subheading 2.3, Primer pair 2)
and the CMV-EGFP cassette from pEGFP-C1 (see Subheading
2.3, Primer pair 3). Use Gibson Assembly to simultaneously
clone the two PCR products into AseI-digested pdCas9-C1 to
create
SL2-sgTelo-MTSa/EGFP/pdCas9-C1.
Generate
SL2-sgTelo-MTSa/BFP/pdCas9-C1 similarly with the
CMV-BFP cassette PCR amplified from pmTagBFP2-C1 plasmid (see Subheading 2.3, Primer pair 4).
3. For centromere labeling, SL2-sgSat-MTSb/BFP/pdCas9-C1
should be similarly generated as SL2-sgTelo-MTSa/BFP/
pdCas9-C1. The U6-sgRNA cassette is PCR amplified from
pU6-SL2-sgSat-MTSb (see Subheading 2.3, Primer pair 5).
3.2 Cellular Delivery
of MBs
Deliver MBs into transfected HEK293 cells by microporation. The
detailed protocol is as follows:
Day 0:
Seed the proper number of HEK293 cells into a 6-well plate
with cell culture medium such that cells will reach 50–70% confluency on day 1 for transfection.
Day 1:
1. Add 200 μL fibronectin into each well of the 8-well chambered
cover glass and incubate at 37
C overnight (see Note 15).
2. Transfect the cells seeded in the 6-well plate with 2 μg of
SL2-sgTelo-MTSa/EGFP/pdCas9-C1 for telomere labeling
or SL2-sgSat-MTSb/BFP/pdCas9-C1 for centromere labeling. Transfect the cells with SL2-sgTelo-MTSa/BFP/
pdCas9-C1 and SL2-sgSat-MTSb/BFP/pdCas9-C1 at a 1:1
ratio (1 μg each) for dual labeling (see Note 16).
Day 2:
1. Aspirate the cell culture medium and incubate the cells with
1 mL pre-warmed PBS at room temperature (RT) for 2 min.
2. Aspirate the PBS and add 0.5 mL of trypsin. Incubate for 1 min
at RT.
3. Aspirate the trypsin (leaving a trace amount). Incubate at 37
C
until all cells are detached from the plate surface.
4. Neutralize the remaining trypsin with ~1 mL of cell culture
medium and gently pipette to resuspend the cells (see
Note 17).
5. Transfer the cell suspension to a 1.5 mL microcentrifuge tube
and pellet the cells by centrifugation at 400 Â g for 5 min at
4
C (see Note 18).
6. Aspirate the medium and gently resuspend the cell pellet with
1 mL of PBS.
Visualizing Genomic Loci by CRISPR/MB
363