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Advanced CRISPR: Part V
Lab Session 29
Advanced CRISPR: Part V Goal: Today you will lyse the cells transfected with CRISPR vectors and PCR amplify the genomic DNA (gDNA) region you intended to target with your sgRNAs.
I.
INTRODUCTION
Not all sgRNAs are equally effective at targeting a gene of interest. Recall that a computer algorithm was used to help design your sgRNAs in order maximize cleavage events within your gene of interest, while minimizing off-target effects. Due to this fact, it is recommended that researchers design and test multiple sgRNAs. This experimental design strategy is similar to that involving RNAi. Even though an sgRNA is intended to create a double-strand break in the gene and lead to an insertion or deletion, one must consider other possible outcomes. Transfection of the CRISPR vector that encodes the sgRNA and Cas9 is never 100% effective. Furthermore, since you transiently transfected cells, the CRISPR vector was only produced in a subpopulation of the cells. In order to maximize transfection efficiency, you were instructed to test multiple amounts of CRISPR vector in your transfections. Even in cells that did receive the vector, the sgRNAs may only target one of the two alleles for your gene of interest. This would lead to DSBs in a heterozygous, not homozygous manner. Thus, CRISPR experiments hinge not only on transfection efficiency, but also cleavage efficiency. A researcher must confirm that an sgRNA created an indel where it was intended to. The CRISPR experiment aimed at knocking out egfp (Lab Sessions 23 and 24) was unique in that a fluorescent, easily detectable gene product was targeted. In most cases, your gene of interest will not be detectable by eye. One must use biochemical techniques to analyze if the targeting was accurate. A quantitative measure of this gDNA cleavage will allow you to determine the most effective sgRNA to move forward with in future CRISPR-mediated gene editing experiments. This lab exercise and the next will serve as primary screens, which are outlined in Fig. 29.1. Today, you will isolate gDNA from your CRISPR vector-transfected cells since it is the gDNA you aimed to disrupt. Then, you will use PCR to amplify a fragment of that gDNA containing the locus where you expect a double-strand break. Remember, this is immediately upstream of a PAM site. In the next lab session, you will denature and reanneal the strands of the PCR product and add an enzyme that detects mismatches in the DNA. A mismatch signals that a DNA strand with an indel has re-annealed with a strand that does not have an indel or has a different indel (see Fig. 29.1 Steps 3 5). By using this assay, you will be able to determine to what extent each of the tested sgRNA conditions resulted in cleavage of a genomic locus.
II.
LABORATORY EXERCISES
The following steps are modified from the GeneArt Genomic Cleavage Detection Kit Manual MAN0009849, 2014. If you are using a different commercially available indel detection assay, please refer to the manufacturer’s instructions. Note to instructors: Cells should be harvested B48 hours post-transfection (Lab Session 28: Advanced CRISPR: Part IV). Longer incubations will result in overly confluent cells. If harvesting as a class cannot occur 48 hours post transfection, instructors should follow the steps under “A: Harvesting Cells” and freeze pellets at 280 C for use on the next lab day. If students are going to continue to maintain CRISPR vector-transfected cells with the goal of creating a clonal population (see Introduction in Lab Session 30) they should harvest and analyze just half of the cells in every transfected well. The other half of the cells in each well can continue to be cultured while this indel detection assay is performed. The instructions in Lab Sessions 29 and 30 are written so that students will stop with the indel detection assay. For that Molecular Biology Techniques. DOI: https://doi.org/10.1016/B978-0-12-815774-9.00029-0 © 2019 Elsevier Inc. All rights reserved.
205
206
PART | V Modulation of Gene Expression
FIGURE 29.1 An overview of the GeneArt Genomic Detection Kit. Lab Session 29, Advanced CRISPR: Part V, will involve Steps 1 and 2. Lab Session 30, Advanced CRISPR: Part VI, will involve Steps 3 5.
reason, all of the cells are harvested for lysates. In addition, lysates can be made from just the pLentiCRISPR-EGFP (negative control for targeting your gene of interest) and each well transfected with pLentiCRISPR-sgRNA (experimentals) for four lysates per group.
A.
Harvesting Cells
1. Obtain your group’s 48-well plate of HEK293 cells that were transfected in Lab Session 28. 2. Using the microscope and 10 3 objective lens, observe and record the morphology and confluency of your cells under white light. Your cells should be extremely confluent at this point. 3. Take your plate of transfected cells to the bench. You will not work with them in a sterile environment, as the cells will be lysed today. Note: if you will also be maintaining these cells in culture to create a clonal population, cells must only be manipulated in the laminar flow hood. 4. Aspirate all of the medium using an in-house vacuum at the bench or a self-contained aspirating pump. Whenever you remove or add liquid to your cells, be sure to do so from the same location to avoid dislodging cells. 5. If using a 48-well plate, gently add B200 µL of 1 3 PBS to the wells: pLentiCRISPR-EGFP and each transfected with pLentiCRISPR-sgRNA. Rock the plate gently. Aspirate. 6. Add B200 µL of pre-warmed 0.25% Trypsin-EDTA to each well and incubate on the bench for B2 to 3 minutes. 7. Add B200 µL of complete media to each well. 8. Pipette up and down to mix and create a cell suspension. 9. Transfer each cell suspension to a labeled microcentrifuge tube. 10. Spin down the cells at 200 3 g for 5 minutes at 4 C. 11. Carefully remove the supernatant with a pipette tip and dispose of all liquid in a waste container containing 10% bleach. Do not attempt to aspirate at this step, because you may lose the pellet of cells!
Lab Session | 29
B.
207
Lysis and Extraction
1. Thaw Cell Lysis Buffer and Protein Degrader at room temperature. You will need to mix 50 µL Cell Lysis Buffer with 2 µL Protein Degrader for each cell lysate you make. Since you have four lysates, calculate the volume of each you will need, being sure to prepare approximately 20% extra to account for pipetting variability. Pipette these into a sterile microcentrifuge tube and pipette up and down to mix. 2. Add 50 µL of Cell Lysis Buffer/Protein Degrader mix to each cell pellet and resuspend the pellet by pipetting up and down. 3. Transfer all of the resuspended cell pellet to a labeled PCR tube. 4. Run this program in a thermal cycler: 68 C 15 minutes 95 C 10 minutes 4 C Hold* *Following completion, immediately proceed to PCR amplification or store at 220 C. Even though this step uses a thermal cycler, PCR is not occurring. The increased temperature helps lyse the cells. Using the instrument is a convenient way to incubate for specific times and temperatures, just like it was used for reverse transcription reactions in Part IV of this book. While the cells are lysing, proceed to pouring agarose gels, as you will need three per group in the next session to confirm: (1) PCR resulted in one specific product from your gene of interest at the correct molecular weight and (2) an indel was detected in a proportion of your cells.
C.
Pouring Agarose Gels
1. Each group will need an agarose gel next week that has enough lanes for: a. one DNA ladder. b. one positive control reaction. c. one lane for each lysate (pLentiCRISPR-EGFP, pLentiCRISPR-sgRNA 3 3). 2. Each group will additionally need enough lanes for: a. one DNA ladder. b. two lanes for the control reaction. c. two lanes for each lysate (eight total). 3. Prepare three separate agarose gels today to accommodate the number of lanes needed next week. You should prepare a 2% agarose gel solution (2 g agarose in every 100 mL TBE). The higher percentage of agarose will create smaller pore sizes to better resolve DNA fragments of similar molecular weight. You will still add 10,000 3 GelRed stock to 1 3 final concentration. 4. Once solidified completely, each group can wrap the entire tray, gel, and comb in plastic wrap and store at 4 C until the next lab session. Your instructor may provide you with different directions for storing gels so that trays and combs can be used by other groups.
D.
PCR
1. Briefly vortex cell lysates once the thermal cycler program is complete. 2. In addition to your lysates, you should also run a positive control that is provided with the kit (five PCR reactions total). This control includes both DNA template and forward and reverse primer mix in one reagent. The gene-specific primers you need to add were designed and ordered in Lab Session 25. Label five new PCR tubes for a control PCR and lysates 1 4. Use labels consistent with what was transfected in each well. 3. The following components will be added to a PCR tube. Create a table in your notebook like Table 29.1 for a master mix containing calculated volumes of everything except lysate (forward and reverse primers, AmpliTaq Gold 360 Master Mix, and water). Vortex the master mix. Distribute 48 µL of that master mix per tube, then add 2 µL of the appropriate cell lysate. Set up the control reaction separately with only control template and primers, AmpliTaq Gold 360 Master Mix, and water. Vortex to mix.
208
PART | V Modulation of Gene Expression
TABLE 29.1 Experimental Set-up for PCR Reagent
Amount Per Reaction (µL)
Cell lysate (serves as DNA template)
2
Forward gene-specific primer (10 µM)
0.5
Reverse gene-specific primer (10 µM)
0.5
Amount for 4 reactions (µL)
Control template and primers (provided in kit)
Amount Per Control Reaction (µL)
1
AmpliTaq Gold 360 Master Mix
25
25
Water
22
24
Total
50
50
4. Keep your PCR tubes on ice until all groups are ready. 5. Run the thermal cycler using the program shown in Table 29.2.
TABLE 29.2 Thermal Cycler Program Stage
Temperature ( C)
Time
Cycles
Enzyme activation
95
10 min
13
Denature
95
30 s
40 3
Anneal
55 60
30 s
Extend
72
30 s
Final extension
72
7 min
13
Hold
4
Indefinitely
13
Note: The above conditions assume that previous primers designed in Lab Session 25, have similar melting temperatures (Tms) around 60 C and result in products of B400 500 bp in length. The annealing temperature should be adjusted based on the primers used.
After the program is complete, your instructor or TA will store your PCR tubes in the 220 C freezer until your next lab session.
DISCUSSION QUESTIONS 1. How does the cell lysis buffer used in the genomic cleavage detection experiment differ from that used in protein purification experiments? What is the purpose of these two buffers? 2. If your sgRNAs failed to target a particular genomic locus, will you get a PCR product? Why?
Advanced CRISPR: Part V Goal: Today you will lyse the cells transfected with CRISPR vectors and PCR amplify the genomic DNA (gDNA) region you intended to target with your sgRNAs.
I.
INTRODUCTION
Not all sgRNAs are equally effective at targeting a gene of interest. Recall that a computer algorithm was used to help design your sgRNAs in order maximize cleavage events within your gene of interest, while minimizing off-target effects. Due to this fact, it is recommended that researchers design and test multiple sgRNAs. This experimental design strategy is similar to that involving RNAi. Even though an sgRNA is intended to create a double-strand break in the gene and lead to an insertion or deletion, one must consider other possible outcomes. Transfection of the CRISPR vector that encodes the sgRNA and Cas9 is never 100% effective. Furthermore, since you transiently transfected cells, the CRISPR vector was only produced in a subpopulation of the cells. In order to maximize transfection efficiency, you were instructed to test multiple amounts of CRISPR vector in your transfections. Even in cells that did receive the vector, the sgRNAs may only target one of the two alleles for your gene of interest. This would lead to DSBs in a heterozygous, not homozygous manner. Thus, CRISPR experiments hinge not only on transfection efficiency, but also cleavage efficiency. A researcher must confirm that an sgRNA created an indel where it was intended to. The CRISPR experiment aimed at knocking out egfp (Lab Sessions 23 and 24) was unique in that a fluorescent, easily detectable gene product was targeted. In most cases, your gene of interest will not be detectable by eye. One must use biochemical techniques to analyze if the targeting was accurate. A quantitative measure of this gDNA cleavage will allow you to determine the most effective sgRNA to move forward with in future CRISPR-mediated gene editing experiments. This lab exercise and the next will serve as primary screens, which are outlined in Fig. 29.1. Today, you will isolate gDNA from your CRISPR vector-transfected cells since it is the gDNA you aimed to disrupt. Then, you will use PCR to amplify a fragment of that gDNA containing the locus where you expect a double-strand break. Remember, this is immediately upstream of a PAM site. In the next lab session, you will denature and reanneal the strands of the PCR product and add an enzyme that detects mismatches in the DNA. A mismatch signals that a DNA strand with an indel has re-annealed with a strand that does not have an indel or has a different indel (see Fig. 29.1 Steps 3 5). By using this assay, you will be able to determine to what extent each of the tested sgRNA conditions resulted in cleavage of a genomic locus.
II.
LABORATORY EXERCISES
The following steps are modified from the GeneArt Genomic Cleavage Detection Kit Manual MAN0009849, 2014. If you are using a different commercially available indel detection assay, please refer to the manufacturer’s instructions. Note to instructors: Cells should be harvested B48 hours post-transfection (Lab Session 28: Advanced CRISPR: Part IV). Longer incubations will result in overly confluent cells. If harvesting as a class cannot occur 48 hours post transfection, instructors should follow the steps under “A: Harvesting Cells” and freeze pellets at 280 C for use on the next lab day. If students are going to continue to maintain CRISPR vector-transfected cells with the goal of creating a clonal population (see Introduction in Lab Session 30) they should harvest and analyze just half of the cells in every transfected well. The other half of the cells in each well can continue to be cultured while this indel detection assay is performed. The instructions in Lab Sessions 29 and 30 are written so that students will stop with the indel detection assay. For that Molecular Biology Techniques. DOI: https://doi.org/10.1016/B978-0-12-815774-9.00029-0 © 2019 Elsevier Inc. All rights reserved.
205
206
PART | V Modulation of Gene Expression
FIGURE 29.1 An overview of the GeneArt Genomic Detection Kit. Lab Session 29, Advanced CRISPR: Part V, will involve Steps 1 and 2. Lab Session 30, Advanced CRISPR: Part VI, will involve Steps 3 5.
reason, all of the cells are harvested for lysates. In addition, lysates can be made from just the pLentiCRISPR-EGFP (negative control for targeting your gene of interest) and each well transfected with pLentiCRISPR-sgRNA (experimentals) for four lysates per group.
A.
Harvesting Cells
1. Obtain your group’s 48-well plate of HEK293 cells that were transfected in Lab Session 28. 2. Using the microscope and 10 3 objective lens, observe and record the morphology and confluency of your cells under white light. Your cells should be extremely confluent at this point. 3. Take your plate of transfected cells to the bench. You will not work with them in a sterile environment, as the cells will be lysed today. Note: if you will also be maintaining these cells in culture to create a clonal population, cells must only be manipulated in the laminar flow hood. 4. Aspirate all of the medium using an in-house vacuum at the bench or a self-contained aspirating pump. Whenever you remove or add liquid to your cells, be sure to do so from the same location to avoid dislodging cells. 5. If using a 48-well plate, gently add B200 µL of 1 3 PBS to the wells: pLentiCRISPR-EGFP and each transfected with pLentiCRISPR-sgRNA. Rock the plate gently. Aspirate. 6. Add B200 µL of pre-warmed 0.25% Trypsin-EDTA to each well and incubate on the bench for B2 to 3 minutes. 7. Add B200 µL of complete media to each well. 8. Pipette up and down to mix and create a cell suspension. 9. Transfer each cell suspension to a labeled microcentrifuge tube. 10. Spin down the cells at 200 3 g for 5 minutes at 4 C. 11. Carefully remove the supernatant with a pipette tip and dispose of all liquid in a waste container containing 10% bleach. Do not attempt to aspirate at this step, because you may lose the pellet of cells!
Lab Session | 29
B.
207
Lysis and Extraction
1. Thaw Cell Lysis Buffer and Protein Degrader at room temperature. You will need to mix 50 µL Cell Lysis Buffer with 2 µL Protein Degrader for each cell lysate you make. Since you have four lysates, calculate the volume of each you will need, being sure to prepare approximately 20% extra to account for pipetting variability. Pipette these into a sterile microcentrifuge tube and pipette up and down to mix. 2. Add 50 µL of Cell Lysis Buffer/Protein Degrader mix to each cell pellet and resuspend the pellet by pipetting up and down. 3. Transfer all of the resuspended cell pellet to a labeled PCR tube. 4. Run this program in a thermal cycler: 68 C 15 minutes 95 C 10 minutes 4 C Hold* *Following completion, immediately proceed to PCR amplification or store at 220 C. Even though this step uses a thermal cycler, PCR is not occurring. The increased temperature helps lyse the cells. Using the instrument is a convenient way to incubate for specific times and temperatures, just like it was used for reverse transcription reactions in Part IV of this book. While the cells are lysing, proceed to pouring agarose gels, as you will need three per group in the next session to confirm: (1) PCR resulted in one specific product from your gene of interest at the correct molecular weight and (2) an indel was detected in a proportion of your cells.
C.
Pouring Agarose Gels
1. Each group will need an agarose gel next week that has enough lanes for: a. one DNA ladder. b. one positive control reaction. c. one lane for each lysate (pLentiCRISPR-EGFP, pLentiCRISPR-sgRNA 3 3). 2. Each group will additionally need enough lanes for: a. one DNA ladder. b. two lanes for the control reaction. c. two lanes for each lysate (eight total). 3. Prepare three separate agarose gels today to accommodate the number of lanes needed next week. You should prepare a 2% agarose gel solution (2 g agarose in every 100 mL TBE). The higher percentage of agarose will create smaller pore sizes to better resolve DNA fragments of similar molecular weight. You will still add 10,000 3 GelRed stock to 1 3 final concentration. 4. Once solidified completely, each group can wrap the entire tray, gel, and comb in plastic wrap and store at 4 C until the next lab session. Your instructor may provide you with different directions for storing gels so that trays and combs can be used by other groups.
D.
PCR
1. Briefly vortex cell lysates once the thermal cycler program is complete. 2. In addition to your lysates, you should also run a positive control that is provided with the kit (five PCR reactions total). This control includes both DNA template and forward and reverse primer mix in one reagent. The gene-specific primers you need to add were designed and ordered in Lab Session 25. Label five new PCR tubes for a control PCR and lysates 1 4. Use labels consistent with what was transfected in each well. 3. The following components will be added to a PCR tube. Create a table in your notebook like Table 29.1 for a master mix containing calculated volumes of everything except lysate (forward and reverse primers, AmpliTaq Gold 360 Master Mix, and water). Vortex the master mix. Distribute 48 µL of that master mix per tube, then add 2 µL of the appropriate cell lysate. Set up the control reaction separately with only control template and primers, AmpliTaq Gold 360 Master Mix, and water. Vortex to mix.
208
PART | V Modulation of Gene Expression
TABLE 29.1 Experimental Set-up for PCR Reagent
Amount Per Reaction (µL)
Cell lysate (serves as DNA template)
2
Forward gene-specific primer (10 µM)
0.5
Reverse gene-specific primer (10 µM)
0.5
Amount for 4 reactions (µL)
Control template and primers (provided in kit)
Amount Per Control Reaction (µL)
1
AmpliTaq Gold 360 Master Mix
25
25
Water
22
24
Total
50
50
4. Keep your PCR tubes on ice until all groups are ready. 5. Run the thermal cycler using the program shown in Table 29.2.
TABLE 29.2 Thermal Cycler Program Stage
Temperature ( C)
Time
Cycles
Enzyme activation
95
10 min
13
Denature
95
30 s
40 3
Anneal
55 60
30 s
Extend
72
30 s
Final extension
72
7 min
13
Hold
4
Indefinitely
13
Note: The above conditions assume that previous primers designed in Lab Session 25, have similar melting temperatures (Tms) around 60 C and result in products of B400 500 bp in length. The annealing temperature should be adjusted based on the primers used.
After the program is complete, your instructor or TA will store your PCR tubes in the 220 C freezer until your next lab session.
DISCUSSION QUESTIONS 1. How does the cell lysis buffer used in the genomic cleavage detection experiment differ from that used in protein purification experiments? What is the purpose of these two buffers? 2. If your sgRNAs failed to target a particular genomic locus, will you get a PCR product? Why?