CRISPR Library Amplification

Introduction

Please refer to our pooled library material pages for amplification protocols that have been developed by the depositor for a specific pooled library available at Addgene. We recommend following the depositor's specific protocol if one is available. If a pooled library does not yet have an amplification protocol, the following protocol can be used as a starting point for CRISPR libraries.

This protocol allows the amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used for screening, barcoding, or other high throughput multiplexed experiments. These pooled plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's genome, for example.

Amplification is usually necessary to produce sufficient quantities of library for experimental applications. Repeated amplifications should be avoided as best as possible due to the inherent possibility of altering the composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the representation of individual plasmids in the pooled library during amplification. This protocol is designed to be as general as possible but note that individual libraries may require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled library from Addgene, these protocols can be found on the Pooled Library page for the specific library.

This protocol assumes familiarity with standard bacterial transformation and basic knowledge of the library being amplified. Please see our protocol on Bacterial Transformation. The generalized pooled library amplification protocol is expected to obtain satisfactory results for libraries up to 200,000 individual plasmids and can be scaled according to the actual number.

Required Quality Control (QC)

Pooled libraries can be challenging and expensive to ensure adequate quality, but this upfront cost will save headaches and expense later.

At a minimum we recommend the use of a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme for digest that will cut the shared plasmid backbone a single time and visualize that digestion on an agarose gel (see restriction digest protocol here). Lentiviral plasmids can recombine between their LTRs resulting in a smaller plasmid containing only the elements required for bacterial propagation (origin of replication and antibiotic selection). This recombination, at a low rate, is not typically a problem as these sequences are not efficiently packaged into lentiviral particles. See below for options if the recombinant band makes up a significant proportion of the DNA pool.

Last Update: August 17, 2023

Workflow Timeline

Day 1:

Transform, recover, set up overnight growth (Estimated time 2-3 hours)
Transformation should be performed at the end of the day to ensure that growth times are limited.

Day 2:

Harvest cells and purify DNA (Estimated time 3-4 hours)
Cells should be harvested first thing in the morning to ensure that outgrowth and competition are limited.

Equipment

• Table top centrifuge
• BioRad Electroporator (MicroPulser TM , Bio-Rad 1652100)

Reagents

• 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1)
  • Alternatives include Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone DNA.
  • The use of electrocompetent cells is essential to ensure high efficiency uptake of plasmid library DNA.
  • This quantity of cells is sufficient for libraries up to 200,000 individual plasmids. For larger libraries scale the number of cells and DNA accordingly.
  Pro-Tip
  Use of extreme high efficiency (1x10¹⁰ cfu/µg) commercially prepared electrocompetent cells is strongly recommended.
• 800 ng pooled library DNA (100 ng plasmid DNA per 25 µL electrocompetent cells)
• Less library DNA can be used but this dramatically increases the chances of individual plasmids being lost from the pool and/or increasing the skewness of the pool.
• 8 electroporation cuvettes (BioRad, Micropulser, 0.1 cm )
• 20 mL SOC recovery media (Lucigen, 80026-1)
• 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular Devices, X602)
  Pro-Tip
  Pour these plates at least one day in advance to allow adequate time to fully gel and to dry slightly. We routinely use 350 mL of LB Agar per bioassay plate.
• 14 mL Vented Falcon Tubes (BD Biosciences, 352059)
• 3X LB Agar + Antibiotic 65 mm Petri dish (VWR, 11019-552)
• 4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663)
• Tips (1000 µL, 200 µL, 10 µL)
• Bacti Cell Spreaders (VWR, 60828-680)
• 5 mL and 10 mL Serological pipettes
• Ice slurry (Ice bucket with ice and water to create slurry)
• 100 mL LB
  • At Addgene we use premixed LB (VWR 101414-072) for convenience but any brand or house- made LB that supports normal growth is expected to work.
• 50 mL Falcon Conical tubes (Fisher, 14-432-22)

Reagent Preparation

1. Prepare, sterilize, and pour all LB Agar + Antibiotic plates.
2. Prewarm 12 mL recovery media at 37 °C (for at least 15 minutes).
3. Prewarm 3X LB Agar + Antibiotic plates at 37 °C.
4. Prewarm 8X LB Agar + Antibiotic Bioassay plates.
5. Prechill Micropulser cuvettes on ice.
6. Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely thawed.
7. Chill a box of 200 µL micropipette tips in a -20 °C freezer.
8. Aliquot 3 mL SOC into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation recovery of cells.
9. Ensure access to autoclaved, sterile reagents for all steps.

Procedure

Day 1

1. Add 200 ng DNA to each 50 µL aliquot of thawed Endura Duos on ice. Flick gently to mix.
2. Electroporate cells (one at a time for a total of eight electroporations):
   • Electroporator Conditions: Bio-Rad Micropulser Ec1 0.1 cm cuvette, 1.8 kV, 1 pulse.
3. Aliquot 25 µL DNA-Endura into pre-chilled cuvette.
4. Pulse.
   Caution
   Electroporation involves the use of high voltages, please use caution when activating pulse and follow all specifications described in the equipment manual.
5. Immediately add 1 mL SOC to cuvette.
6. Remove all liquid from cuvette and add to 14 mL vented Falcon Tube containing 3 mL SOC.
7. Repeat for each of the 25 µl aliquots of cell/DNA mixture used (8 in the described protocol) using a new vented falcon tube for every two transformations. Each of the four 14 mL Vented Falcon Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations).
   Pro-Tip
   Do not pipette repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette.
8. Shake four 14 mL Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour.
9. After the 1 hour shaking period, pool and gently mix the four tubes.
10. Perform sequential 1:100 dilutions of the cells (add 10 µL of the pool to 990 µL LB then perform a second and then third 1:100 dilution). Plate 100 µL of each dilution onto a prewarmed Petri dish.
11. Incubate plates at 30 ℃ overnight.
12. Plate 2.5 mL of the transformed cells on each of the eight bioassay plates (two plates per tube). Distribute evenly with a sterile spreader until all liquid has been absorbed by the agar. This usually takes 1-2 minutes.
    Critical
    Be careful not to rip or shred the agar. Do so by gentle spreading. Some spreaders have a sharp edge that can scrape plates more abrasively at a certain angle.
13. Incubate plates upside down at 30 ℃ overnight.
    Critical
    Ensure at this stage that no unabsorbed media drips onto the lid. Let plates remain agar side up until dried before overnight incubation if needed.
14. Place 100 mL sterile LB at 4 ℃.

Day 2 (morning)

15. Before beginning, prechill at least four conical tubes on ice and ensure access to sterile scraper and cold LB.
    Pro-Tip
    Prepare one to two more conical tubes on ice in case you need to spread out the harvested cells further than four Maxipreps worth.
16. Count colonies on the most dilute Petri dish.
    • Total colony yield = count x 100 x 100 x 100 ÷ 0.1
    • This number should be at least 1000X greater than number of perturbations in library.
      Example: At least 10 colonies on the most diluted plate for 1000x coverage of a library of 100,000 plasmids.
    • Colonies may appear small and require extra incubation time in order to be enumerated accurately.
    • Frequently the number of colonies can be too great to count. Ideally, dilutions would have been sufficient to enumerate single colonies but as long as one can ensure more than the required colonies are present. If colonies are not present on the most dilute plate, count the second plate (ie. the 1:10,000 dilution).
17. After 12-18 hours of growth, use spreader and cold LB to scrape bioassay plates and remove bacteria.
18. Use one scraper for all plates.
19. Use two 10 mL pipettes (one for dispensing cold LB and one for removing LB containing bacteria from plate).
    Pro-Tip
    Scraped bacteria in LB can clog the pipette either mix gently up and down avoiding introducing bubbles or pour off plates into conical tubes as needed.
20. Add 10 mL cold LB to each plate for each scrape and use spreader to scrape plates.
    Pro-Tip
    Pushing motion is better than pulling motion Take care not to split or gouge agar during the scraping process.
21. Add each scrape into a 50 mL conical tube on ice pooling the scrapings of two plates into each tube. Keep each tube on ice while scraping.
22. Repeat addition of 10 mL cold LB and scrape for each plate until agar is clear (all bacteria have been removed). This should produce 25 mL cold LB-bacteria per plate.
23. Centrifuge tubes (4000 G, 4 ℃, 15 minutes) to pellet bacteria.
24. Decant LB and weigh pellet. The total weight of each pellet should be ~1-2 g.
    Pro-Tip
    Make sure to weigh the empty tube beforehand! If you’ve already gone too far, weigh an identical empty tube. It will be close enough for the purpose of a Maxiprep.
25. Purify plasmid DNA using the Qiagen HiSpeed Maxi Kit (one conical is its own Maxiprep).
    Critical
    Do not freeze pellets for later purification. Immediately purify them! Commercial Maxipreps rely on incremental, ordered cell lysis. E. coli cells are subject to lysis by freeze-thaw if not suspended in cryoprotectant like Glycerol or DMSO solutions.
26. Quantify the individual Maxipreps by Nanodrop, Picogreen, or Qubit.
    • Each different DNA measurement type can report slightly different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than Nanodrop, although most protocols have been designed with Nanodrop as the quantification method.
27. If all Maxipreps appear to contain sufficient DNA for use, pool samples, and continue with analysis.
    • “Sufficient” DNA will vary upon intended use. Typically yields range from several hundred micrograms to a milligram of total plasmid DNA.

Tips and Troubleshooting

What do I do if my transformation efficiency is not high enough?

Repeat the experiment with more cells, more DNA, gentler practices, and colder reagents (not including the recovery media!). Do not proceed with Maxipreps or NGS until adequate transformation efficiency is obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide adequate transformation efficiency! Ensure that no arcing is taking place during electroporation. Arcing would manifest as a loud pop often accompanied by some light in the cuvette during the electroporation step.

I got a heavy recombined band by digest.

Recombination is thought to be in part related to selective pressure. These recombined plasmids are much smaller and are a smaller nutritional burden on the bacteria. Bacteria that contain a recombinant plasmid likely grow faster in the closed system of a crowded plate or culture. Try restricting growth to the bare minimum by growing at 30 ℃, limiting growth time further, or potentially spreading less bacteria on a given plate (example: by increasing the total number of bioassay plates used). Scaling up the experiment using a Gigaprep can be used to obtain sufficient intact pooled library even in the face of a recombined fraction but care must be taken to perform adequate NGS based QC to ensure no change in representation compared to the pre-amplified stock. A last option is to perform a gel extraction of either the original sample or the amplified sample, followed by reamplification of the DNA, but please note that NGS should be performed to ensure representation is maintained.

Maxipreps - Less is more:

Do not overload the Maxipreps as yield can dramatically plateau and sometimes fall off entirely if the column or reagents are significantly overloaded. If you find that your pellet outstrips the capacity listed in the Maxiprep protocol, scale the reagent volume and column number as needed. The use of Mega or Gigapreps is acceptable when scaling up or reducing the number of tandem purifications. Consult the manufacturers handbooks for appropriate volumes and numbers of tandem purifications.

Visit our Help Center to get details on how to process your Addgene pooled library