CRISPR droplet sequencing

Part A: Pooled, amplification-free cloning of gRNA libraries

 Validation of the CROPseq-Guide-Puro plasmid preparation

Commercially available plasmid DNA kit can be purchased.  Perform extraction and validation of the plasmid DNA by restriction digestion and Sanger sequencing.

Preparation of CROPseq-Guide-Puro vector backbone

 Plasmid digestion

Digest CROPseq-Guide-Puro plasmid and gel-purify the 8,333 bp fragment as indicated. Once prepared, the backbone fragment can be frozen at -20 °C and re-used for cloning further gRNA libraries.

Vector digestion mix

Vector Digestion mix Per reaction  Master mix for # of digestion
Component Volume Amount 4 6 8 10 12
CROP seq-Guide-PRO 5 mg
NEB Buffer 1.1 2.5 ml  1x 10 15 20 25 30
BsmBI 2 ml 20 U 8 12 16 20 24
Nuclease free water To 25 ml

Per reaction Master mix for # of digestions

Distribute 25 μl of vector digestion mix per well into a tube strip, incubate: 1 hour at 55 °C (digestion), 20 min at 80 °C (inactivation of the enzyme), hold at 10 °C.

 UV-free gel extraction 

  1. Prepare required number of 0.8% agarose gels with TAE buffer containing 1.8 μg/ml Crystal Violet:

Example for one gel: 0.4 g agarose in 50 ml TAE, plus 40 μl of 2 mg/ml Crystal Violet Solution

Important: use a thin comb for more intense bands, TBE is not compatible with this kit

  1. Load 8 μg of DNA ladder in 25 μl of water, mixed with 5 μl of 6x Crystal Violet Loading Dye
  2. Load 25 μl of each plasmid digestion, mixed with 5 μl of 6x Crystal Violet Loading Dye
  3. Run at 80 V and observe the separation of bands with the naked eye
  4. Once the bands are sufficiently resolved, stop and isolate 8,333 bp fragment with gel-cutting tips
  5. Transfer gel slice to 1.5 ml tube, weigh to estimate the agarose volume (assuming that 1 mg ~ 1 μl)
  6. Add 2.5 volumes of Sodium Iodide Solution
  7. Incubate at 42 °C with occasional vortexing, until the agarose is completely dissolved
  8. Add 1.5 volumes of Binding Buffer, mix and load onto SNAP Purification Column
  9. Centrifuge at 3,000 rcf for 30 seconds
  10. Pour the flow through back onto the column and repeat the centrifugation, for a total of three times
  11. After the last centrifugation step, discard the flow through
  12. Wash two times with 400 μl of 1x Final Wash Buffer, discard flow through after second centrifugation
  13. Centrifuge column for 1 min at > 10,000 rcf to remove residual wash buffer
  14. Transfer the column to a fresh 1.5 ml tube and elute with 40 μl of EB buffer (10,000 rcf for 1 min). Important: incubate for at least 1 min to allow absorption into the column
  15. Transfer the eluate back to the column, and elute a second time as described above
  16. Measure 1 μl of the eluate in a Qubit HS assay to determine the concentration of dsDNA

 <Safe Stopping Point> The backbone fragment can be stored at -20 °C and re-used.

<Troubleshooting> If required, perform ethanol precipitation to further concentrate the DNA: pool multiple gel-purification reactions, add 1/10 volume of 3M sodium acetate, pH 5.5 and 2.5 volumes of 100% ice cold ethanol. Precipitate DNA for 30 min at -80 °C, then spin for 20 min at 20,000 rcf at 4 °C to pellet. Wash pellet two times with room temperature 70 % ethanol, leave to dry (take care not to over-dry the pellet) and elute in a smaller volume of EB buffer.

Assembly of gRNA-encoding ssDNA oligonucleotides into the vector backbone using Gibson Assembly

 Design gRNAs

Order as separate ssDNA oligos, with 18 and 35 bases of homology to the hU6 promoter

and gRNA backbone for Gibson Assembly. The 5’ prefix also adds a G as a constant first base for more efficient transcription from the hU6 promoter. Examples for positive controls (targeting essential genes) and negative controls (non-targeting) can be found in the original publication of CROP-seq.

Gibson Assembly

Dilute gRNA-encoding ssDNA oligos to 100 μM with EB buffer. Pool all oligos contained in the library (including positive and negative controls) in equal amounts. Dilute the oligo pool 1:1000 in EB buffer, resulting in a molarity of 100 nM. Set up Gibson assembly reactions for each library and the included NEBuilder HiFi positive control, as described below.

Assembly of gRNA-encoding ssDNA oligos into the vector backbone by Gibson Assembly
Per reaction
 

Component

 Volume Amount
CROPseq-Guide-Puro backbone fragment (8,333 bp) 11 fmoles
Library ssDNA oligos, pooled (100 nM)     2.0 μl 200 fmoles
NEBuilder HiFi DNA Assembly Master Mix     10 μl 1x
Nuclease-free water to 20 μl

 

  1. Incubate at 50 °C for 1 hour, then dialyze the reaction against water:

Fill a 6 cm cell culture dish with HPLC-quality water, place membrane filter on top

Pipette 20 μl of the Gibson Assembly reaction and let float on top of the filter for 30 min

<Note> This removes salts and makes the electroporation more efficient

  1. About 10 μl of the Gibson Assembly reaction can be recovered after the filtration

Electroporation of Lucigen Endura cells

 Preparations

  • Prepare mixture of ice and water
  • Thaw one vial of Lucigen Endura electrocompetent cells on ice (takes about 20 min, sufficient for cloning one gRNA library with two electroporations)
  • Pre-chill two 1 mm electroporation cuvettes on ice
  • Place two empty 2 ml tubes on ice
  • Pre-warm Lucigen recovery medium at 37 °C
  • Pre-warm two 25 x 25 cm LB-agar plates containing 100 μg/ml carbenicillin at room temperature

Electroporation

  • Aliquot 25 μl of bacteria into pre-chilled 2 ml tubes, and keep on ice
  • Add 10 μl of de-salted Gibson Assembly to the bacteria and gently tap the tube to mix
  • Transfer to electroporation cuvette, tap to bring contents to the bottom, check for air bubbles
  • Prepare P1000 pipette filled with 1 ml of Lucigen recovery medium
  • Electroporate with the following settings: 25 μF, 200 Ω, 1.5 kV
  • Immediately after the pulse, add the prepared Lucigen recovery medium
  • Gently pipet up and down two times, then transfer to round-bottom tubes
  • Take a note of the time constant for the electroporation (should be ~4.5 msec)
  • Incubate for 1 hour at 37 °C for recovery
  • Prepare a 1:100 dilution (10 μl bacteria plus 990 μl SOC medium), and plate 100 μl on a 10 cm LB agar dish
  • Plate the remaining bacteria onto the 25 x 25 cm LB-agar plate
  • Incubate for 20 hours at 32 °C

 Plasmid preparation

  1. The next day, count colonies on the 1:1000 dilution plate and calculate the library coverage as follows:

(number of colonies x 1000) / (number of gRNAs in library)

  1. Retrieve bacteria from plates as follows:
  • Add 20 ml of pre-warmed LB medium to the plate, scrape off bacteria, and transfer to 50 ml tube
  • Add another 30 ml of pre-warmed LB medium, tilt plate and transfer to 50 ml tube
  • Spin at 4,700 rcf for 15 min to collect a bacterial pellet and discard supernatant
  1. Proceed with plasmid extraction using the Endo-Free Plasmid Mega kit according to the manufacturer’s instructions

Next-generation sequencing of gRNA sequences for library quality control or pooled screens

gRNA sequences are amplified from a CROPseq-Guide-Puro plasmid library or from the gDNA of transduced cells (pooled screen) in a single PCR reaction with specially designed primers. This step adds all sequences required for compatibility with Illumina sequencers. In addition, i7 barcodes are provided for sample multiplexing, and stagger sequences can increase library complexity for sequencing.

To retain a good library representation and reduce bias, multiple PCR reactions should be performed for each gRNA plasmid library or gDNA preparation. The number ultimately depends on the size of the gRNA library, but we mostly use four parallel PCR reactions. These reactions can use the same i7 barcode, so that they are demultiplexed into the same file during data processing.

 

qPCR amplification of gRNAs from the plasmid library or gDNA of transduced cells
Per reaction
Component volume Amount
CROPseq-Guide-Puro plasmid library 10 ng
or gDNA from a pooled screen or 100 ng
Q5 Hot Start High-Fidelity 2x Master Mix 25 μl 1x
CROPseq_libQC_i5 primer (10 μM) 2.5 μl 500 nM
CROPseq_libQC_i7 primer (10 μM) 2.5 μl 500 nM
Optional: SYBR Green, 100x 0.5 μl 1x
Nuclease-free water to 50 μl

 

By adding SYBR Green, the reactions can be run in a qPCR machine and stopped just before reaching the plateau phase. This ensures that the optimal number of enrichment cycles is used. When running multiple samples, the machine can be paused during the denaturation phase. Pierce the foil to remove finished reactions, and resume the amplification of the remaining samples.

Incubate as follows:

  • 98 °C for 30 sec (initial denaturation)
  • 40 cycles { 98 °C for 10 sec 72 °C for 45 sec }
  • stop qPCR reactions just before they reach the plateau phase

Clean PCR products with a 2.0x AMPure XP bead clean-up

Quality control on is done on High Sensitivity DNA chip should show a single band at 282 bp

Measure the dsDNA concentration in a Qubit HS assay

Dilute library to 4 nM with EB buffer + 0.1% Tween based on size and concentration measurements

Sequence on an Sequencing machine, using a 150-cycle v3 flowcell with a read configuration of 167 bases (read 1) and 8 bases (i7 index).

Lentivirus production and cell culture for CROP-seq screens

Production of lentiviral particles

Prerequisites

Plasmid pool of gRNA library cloned as described in part A, and validated by NGS Plasmid preparations of the 3rd generation lentiviral packaging plasmids pMDLg/pRRE , pRSV-Rev, and pMD2.G.

Cell line(s) stably expressing Cas9 after transduction with lentiCas9-Blast.  The lentivirus

production and transduction protocols below can be used to produce this cell line

<Note> The required scale of the lentivirus production depends on the size of the gRNA library and the planned experiments. For most applications 10 cm dishes, or even 6-well plates should suffice, due to the relatively high titers achieved with the CROPseq-Guide-Puro construct. Numbers for both formats are provided below (color-coded). <Important> Dispose of lentivirus-containing materials according to your institute’s biosafety guidelines. Persons performing experiments with lentivirus must be properly trained.

 Prepare lentivirus packaging medium: Opti-MEM I, 5% FCS, 200 μM sodium pyruvate, no antibiotics.

Seed HEK293T cells at 7 million cells per 10 cm dish in 12 ml of lentivirus packaging medium, or at 1.2  million cells per well on a 6-well plate in 2 ml of lentivirus packaging medium

The next morning proceed with the transfection

Prepare transfection mixtures A and B as indicated below

Transfection mix Component Volume Amount Volume Amount

Transfection of HEK293T cells for lentivirus production
10 cm dish 6-well plate
Transfection mix Component Volume Amount Volume Amount
A CROPseq-Guide-Puro gRNA library, plasmid pool 10.2 μg 1.7 μg
pMDLg/pRRE 5.4 μg 0.9 μg
pRSV-Rev 5.4 μg 0.9 μg
pMD2.G 5.4 μg 0.9 μg
Opti-MEM I 1.5 ml 250 μl
P3000 enhancer reagent 36 μl 6 μl
B Opti-MEM I 1.5 ml 250 μl
Lipofectamine 3000 reagent 42 μl 7 μl

 

Prepare lipid-DNA complexes by combining transfection mixes A and B, and incubating for 20 min at room temperature

In the meantime, remove 6 ml, or 1 ml of medium from the HEK293T cells

Add 3 ml or 500 μl of lipid-DNA complexes to the HEK293T cells, and gently agitate

After 6 h of incubation at 37 °C and 5% CO2 exchange medium for 12 ml or 2 ml of fresh lentivirus packaging medium

Collect lentivirus-containing medium after 24 h, store at 4 °C, add 12 ml or 2 ml of fresh lentivirus packaging medium

Collect lentivirus-containing medium after 48 h, pool with supernatant obtained the previous day and filter through a 0.45 μm filter to remove cells

Aliquot lentivirus preparation into cryotubes and freeze at -80 °C

Proceed with titration of the lentivirus preparation and pooled screening

Optional – Titration of lentivirus preparations

<Note> To save time, transductions for pooled screens can be performed with variable volumes of the virus preparation. All transduction conditions are then selected for 2 days, and the condition with the desired MOI can be chosen for the screen.

 

  • Seed HEK293T cells onto 24-well plates at 50,000 cells per well in 500 μl of complete culture medium (DMEM, 10% FCS, penicillin-streptomycin)
  • Grow overnight at 37 °C and 5% CO2 so that cells reach 30% to 50% confluence
  • Exchange culture medium for 450 μl complete culture medium, supplemented with 8 μg/ml polybrene
  • Thaw one vial of the lentivirus preparation from storage at -80 °C
  • In a 96-well plate, prepare a 1:5 dilution series of the lentivirus preparation in complete culture medium with polybrene, ranging over 10 wells (dilution factors from 1:5 to 1:9,765,625)
  • Test the original stock, and each dilution in duplicate, by adding 50 μl per well to the 24-well plate with HEK293T cells (this requires 2 x (1 + 10) = 22 wells).
  • The remaining two wells serve as untransduced controls. Add 50 μl complete culture medium containing polybrene to test for toxicity of polybrene to cells (possible for some cell types).
  • 24 h post transduction exchange the medium for 500 μl complete culture medium without polybrene
  • 48 h post transduction start selection with puromycin and renew selective medium every 2-3 days. The table below provides common antibiotic concentrations we use for puromycin selection, but ultimately the concentration will depend on the cell line used.
Examples of selection conditions used in our lab
Cell line c(Puromycin) [μg/ml]

to select for gRNA

c(Blasticidin) [μg/ml]

to select for Cas9

K562 2 30
Jurkat 2 25
MV4-11 1 20
KBM7 0.5 20
HEK293T 2.25 22.5

 

  • As soon as cells in the untransduced controls are dead, remove medium and wash once with 1x PBS Stain colonies for 15 min in a solution of 1% (w/v) crystal violet in 10% ethanol
  • Wash several more times with 1x PBS, count colonies for two dilution factors, and two replicates each
  • Calculate the virus titer (transducing units/ml) as (average number of resistant colonies) x (dilution factor of well) x (1,000 μl/50 μl)
  • Example for a typical gRNA library: 56 and 66 colonies were counted for the well with dilution factor 15,625 ((56 + 66) / 2) x (15,625) x (1,000 μl/50 μl) = 1.9 x 107 TU/ml

 Transduction of suspension cells for pooled CROP-seq screens

  • Seed suspension cells in 6-well plates at 5 million cells per well in 2 ml of complete culture medium (depends on cell line), supplemented with 8 μg/ml polybrene and blasticidin (we recommend to keep up selection for lentiCas9-Blast throughout the screen, as it is easily silenced; please refer to the table above for suggestions on blasticidin concentrations for various cell lines).
  • Option 1 (virus titer was measured): Add calculated volume of lentivirus preparation for the desired MOI to one well, use a second well as untransduced control.
  • Option 2 (virus titer unknown): Transduce five wells with different volumes of the lentivirus preparation (e.g. 250 μl or 50 μl of the original stock, or 50 μl of 1:3, 1:6 and 1:12 dilutions). The sixth well is left
  • Immediately after addition of lentiviral particles, centrifuge the plate for 45 min at 1,200 rcf and 37 °C (spinfection), followed by over night incubation at 37 °C and 5% CO2.
  • 24 h post transduction, pellet cells in 15 ml tubes, remove supernatant. Transfer cells to T75 cell culture flasks in 30 ml of fresh complete culture medium supplemented with blasticidin (still only selecting for lentiCas9-Blast). 48 h post transduction, start selection for the gRNA library by adding puromycin directly to the flask. Renew selective medium containing blasticidin and puromycin every 2-3 days for a total of 7- 10 days to allow for efficient genome editing. If option 2 was chosen, select the flask with about 30% surviving cells after 2 days of puromycin selection. 

Transduction of adherent cells for pooled CROP-seq screens 

  • Seed adherent cells in 15 cm dishes at 5 million cells per plate in 20 ml of complete culture medium (depends on cell line) supplemented with 8 μg/ml polybrene and blasticidin (we recommend to keep up selection for lentiCas9-Blast throughout the screen, as it is easily silenced;
  • Option 1 (virus titer was measured): Add calculated volume of lentivirus preparation for the desired MOI to one plate, use a second plate as untransduced control.
  • Option 2 (virus titer unknown): Transduce five plates with different volumes of the lentivirus preparation (e.g. 250 μl or 50 μl of the original stock, or 50 μl of 1:3, 1:6 and 1:12 dilutions). A sixth plate is used as the untransduced control.
  • After addition of lentiviral particles, swirl the plate gently to mix and incubate over night at 37 °C and 5% CO2. 24 hours post transduction, exchange medium for fresh complete culture medium supplemented with blasticidin (selecting for lentiCas9-Blast). At 48 h post transduction, start selection for the gRNA library by adding puromycin directly to the plate. Renew selective medium every 2-3 days for a total duration of 7-10 days to allow for efficient editing. If option 2 was chosen, select the plate with about 30% surviving cells after 2 days of puromycin selection.