sgRNA library re-amplification in liquid culture

Erik Haussner, Michael Böttcher

Published: 2023-10-29 DOI: 10.17504/protocols.io.n92ldmr7xl5b/v1

Abstract

In this protocol, we describe a stepwise procedure for the re-amplification of sgRNA libraries in liquid culture. In our hands, this protocol works reliably to amplify pre-cloned sgRNA libraries (e.g. order from Addgene) in a way that preserves the distribution of library elements.

Before start

Keep your original stock safe and aliquoted. For large plasmids with complementary sequences such as LTR sites, keep in mind that repeated reamplification from an already reamplified stock may lead to an accumulation of recombined plasmids and a poorer distribution of library elements.

Steps

Library transformation

Prepare Sample and for electroporation.

1.1.

Thaw

0h 5m 0s on ice.

1.2.

Pre-cool by placing it on ice.

1.3.

Pre-warm at 37°C .

Add 100ng Sample into 25µL , carefully mix by pipetting up and down.

Add 25µL of the plasmid/cell mix into a cuvette, electroporate at 1.2 kV, 25 uF and 200 ohm or alternative setting (see note below). Directly after electroporation, add 1mL of pre-warmed .

Note

The electroporator setting may vary from model to model and should be checked along with the test plasmids provided in the kit of the STBL4 cells.

Safety information

Make sure that any water or ice residue is removed from the cuvettes before inserting them into the electroporator to avoid arching.

Library recovery

After electroporation, add the 1mL resuspended cells in a 14 ml culture tube and incubate the cells in a thermoshaker 600rpm .

Note

In general, an incubation temperature of 37°C is optimal for cell recovery. Since E. coli tend to recombine plasmids with complementary sequences (e.g. LTRs), recovery temperature can be reduced to 30°C. This may however, result in a lower total number of recovered cells.

Determination of transformation efficiency

Use a small fraction of your cells to determine the electroporation efficiency of the reamplification.

Note

In this step, much depends on the size of the particular plasmid and the number of elements in the library. Therefore, the dilution factor must be chosen based on properties of the library and the scale of the electroporation. Smaller plasmids yield significantly more colonies than large ones, and an upscaled plasmid input at the electroporation step may result in higher dilutions being required to achieve a countable number on the respective agar plates after plating.

5.1.

For 1:10,000 dilution:

Prepare . Take 10µL of recovery culture and dilute in 990µL of (1:100 dilution). Take 100µL of 1:100 dilution and dilute in 900µL of (1:1,000) and plate 100µL on (1:10,000 dilution).

5.2.

For 1:1,000,000 dilution:

Take 10µL of the 1:1,000 dilution and dilute in 990µL of and plate 100 uL on a pre-warmed (1:1,000,000 dilution).

Note

When preparing the dilution series, always mix stock solutions well by flicking the tube before diluting, to resuspend sedimented cells. Distribute the plated cells evenly over the plate by e.g. using glas beads.

Place the plates in an incubator at 37°C .

Library extraction and quality control

Use rest of recovery to inoculate up to 500mL of with an added selection marker specific antibiotic like ampicillin in an Erlenmeyer flask for culture.600rpm

On the next day, check for overall coverage via colony counting on . The overall colony count should be 1000x the element number of your library.

Note

Below we provide a simplified example for how to determine transformation coverage. Example calculation of coverage: On the 1:10,000 dilution plate we count 100 colonies. This gives us 100*10,000 = 1,000,000 total colonies. This total colony number is divided by the number of elements (e.g. sgRNAs) in the respective library. For a library the size of 1,000 sgRNAs, the coverage would 1,000,000/1,000=1,000x . For larger libraries, e.g. the size of the genome-wide Brunello library (80,000 sgRNAs), we would count the 1:1,000,000 dilution plate. In this case, 80 counted colonies would mean 80,000,000 total colonies which divided by the library size (80,000 sgRNAs) would again return a transformation coverage of 1,000x.

Library preparation and QC

Follow the protocol instructions of the for transfection-grade plasmid DNA for Midi Prep. Follow the protocol instructions of the for transfection-grade plasmid DNA for Midi Prep.

10.

Determine your final Sample concentration via NanoDrop or Qubit measurement.

Equipment

Value	Label
Qubit 2.0 Fluorometer instrument	BRAND
Q33226	SKU
with Qubit RNA HS Assays	SPECIFICATIONS

Equipment

Value	Label
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer	NAME
UV-Vis Spectrophotometer	TYPE
Thermo Scientific	BRAND
ND-ONE-W	SKU
https://www.thermofisher.com/us/en/home.html	LINK

11.

Send a sample of your reamplified Sample for Sanger sequencing.

Below we show an example chromatogram of an expected sequencing result. We recommend using sequencing primers 50-100 nt upstream of the sgRNA region. You should see clean traces up- and downstream of the SPACER region, and a noisy 20 nt signal in the SPACER region, due to the sgRNA diversity in your library.

Note

! To validate the distribution of elements in your library, we strongly recommend performing next generation sequencing of your plasmid pool before proceeding with downstream experiments. To do so, follow the NGS protocol provided with your library, using the plasmid pool as template, instead of the genomic DNA (as you would in CRISPR screens).

QC: Plasmid recombination check

12.

Since sgRNA library plasmids can recombine during E.coli re-amplification, it is recommended to check for recombination via linearization of 200ng of your reamplified Sample via a restriction digest within the backbone of your library vector.

13.

Prepare a by melting 1g of in 100mL

14.

Let the required amount for casting cool down till it is approxametly 50°C and add 1µL of per ml of melted

15.

Pour the warm, still liquid gel into an electroporation chamber and wait until it has cooled down.

16.

Mix your liearized reamplified Sample with and prepare for gel loading.

17.

Add your linearized reamplified Sample alongside with the prepared onto the gel and run it for 1h 0m 0s at 120 V.

18.

Check the plasmid size on your gel using UV excitation.

Citation

In the best case, only one band will be present, corresponding to the size of the respective vector. In case of recombination, one or more additional bands will be present. For optimal downstream results, the band of the intact vector should be dominant.

sgRNA library re-amplification in liquid culture

Abstract

Before start

Steps

Library transformation

Library recovery

Determination of transformation efficiency

Library extraction and quality control

Library preparation and QC

QC: Plasmid recombination check

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