CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells

Overview

This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.

Protocol

1. gRNA Design for the CRISPR-concatemer Vector

NOTE: The aim of this section is to explain how to opt for the best targeting strategy and how to design gRNAs containing specific overhangs for the CRISPR-concatemer vector.

1. Design gRNAs against the genes of interest using a CRISPR gRNA design tool of choice. See the Table of Materials for an example.
   NOTE: When targeting a pair of paralogous genes, although it is possible to design one gRNA per gene, it is advisable to design two gRNAs per gene to increase the chances of achieving a double knockout (Figure 1).
2. Make sure the gRNAs do not contain the BbsI recognition site by using a restriction mapping tool (see the Table of Materials for an example).
3. Add specific CRISPR-concatemer vector overhangs to each oligo, as shown in Table 1.

2. Cloning of gRNAs into the CRISPR-concatemer Vector

1. Phosphorylation and annealing of oligos
   NOTE: This step illustrates how to anneal top and bottom strands for each gRNA oligo and how to phosphorylate their ends in a single reaction.
   1. Prepare the reaction mixture for phosphorylating oligos and annealing top and bottom strands on ice, per the instructions below.
      NOTE: All oligos can be pooled into one reaction; for example, in the case of a 4 gRNA-concatemer vector, pool together 8 oligos.
   2. For 3 concatemers, use 3.0 μL gRNA top strand (1.0 μL from each gRNA; 10 μM, 1 μL/gRNA), 3.0 μL gRNA bottom strand (1.0 μL from each gRNA; 10 μM, 1 μL/gRNA), 2.0 μL T4 DNA ligase buffer (10x), 1.0 μL T4 PNK, and add H₂O up to total volume of 20.0 μL.
   3. Mix well by pipetting and run this in a thermocycler using the following settings: 37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 0.3 °C/min, hold at 4 °C.
2. BbsI shuffling reaction
   NOTE: In this section, the pre-annealed gRNA oligos are incorporated into the appropriate position of the concatemer vector in one step by alternating cycles of digestion and ligation.
   1. Dilute the reaction mixture 1:100 in DNase/RNase-free water to generate 3 and 4 gRNA-concatemer vectors.
      NOTE: When cloning 2 gRNA-concatemers this step is not needed.
   2. Assemble the BbsI shuffling reaction on ice, per the instructions below. Include a negative control that only contains the vector.
   3. Use 100 ng CRISPR-concatemer vector, 10.0 μL oligo mixture, 1.0 μL BSA-containing restriction enzyme buffer (10x), 1.0 μL DTT (10 mM), 1.0 μL ATP (10 mM), 1.0 μL BbsI, 1.0 μL T7 ligase, and H₂O up to total volume of 20.0 μL.
   4. Mix well by pipetting and run this in a thermocycler using the following settings: Run 50 cycles for cloning 3 and 4 gRNA-concatemers and 25 cycles for 2 gRNA-concatemers, both at 37 °C for 5 min, 21 °C for 5 min, hold at 37 °C for 15 min, then 4 °C forever.
3. Exonuclease treatment
   NOTE: This step is highly recommended as it increases the efficiency of cloning by removing any traces of linearized DNA.
   1. Treat the BbsI shuffling reaction with a DNA exonuclease (see Table of Materials) as follows.
   2. Take 11.0 μL ligation mix from the previous step (2.2.3), add 1.5 μL exonuclease buffer (10x), 1.5 μL ATP (10 mM), 1.0 μL DNA exonuclease, and bring up total volume to 15.0 μL with water. Incubate at 37 °C for 30 min followed by 70 °C for 30 min.
      NOTE: This step removes any residual linearized DNA in the mixture and so increases the cloning efficiency.
   3. Use 2 μL of the reaction mixture for transformation into chemically competent E. coli bacteria by heat shock.
      ​NOTE: Alternatively, the reaction can be stored for up to one week at -20 °C.

3. Transfection of Intestinal Organoids by Electroporation

NOTE: Please note that this procedure is based on the protocol published by Fujii et al. in 2015, with adaptation for mouse small intestinal organoid cultures.

1. Pre-electroporation
   NOTE: This section describes how to prepare the mouse intestinal organoids prior to electroporation by removing all antibiotics and conditioned media from their culture medium. This will prevent possible toxic effects during electroporation.
   1. On day 0 of the transfection procedure, split organoids in a 1:2 ratio.
      ​NOTE: Intestinal organoid cultures can be obtained by performing crypt isolation according to previously established protocols. Please refer to Table 2 for all media compositions.
      1. When splitting organoids for electroporation, seed a minimum of 6 wells of a 48-well plate per transfection.
      2. Seed the organoids in 20 μL-basement matrix drops and grow them in WENR + Nic medium (Wnt + EGF + Noggin + R-spondin + Nicotinamide) at 37 °C, 5% CO₂ in a humidified incubator (as previously described).
   2. On day 2, change medium by replacing WENR+Nic with 250 μL of EN (EGF + Noggin) + CHIR99021 (Glycogen Synthase Kinase-3 inhibitor) + Y-27632 (ROCK inhibitor), without antibiotics (see Table 2).
      ​NOTE: In all the steps, the quantity of medium added to each well of a 48-well plate is 250 μL.
   3. On day 3, change the organoid medium to EN + CHIR99021 + Y-27632 + 1.25% v/v Dimethyl sulfoxide (DMSO), without antibiotics.
2. Preparation of the cells
   NOTE: Here we describe how to fragment organoids into small cell clusters by mechanical and chemical dissociation. These steps are critical to the success of the procedure.
   1. On day 4, disrupt the basement matrix domes using a 1 mL pipette tip and transfer organoids to a 1.5 mL tube. Pool contents of four wells of a 48-well plate into a tube.
   2. Mechanically break organoids into small fragments by pipetting up and down with a P200 pipette approximately 200 times. Centrifuge at room temperature, 5 min at 600 x g.
   3. Remove the medium and resuspend the pellet in 1 mL of a cell culture grade recombinant protease (see table of materials). Incubate at 37 °C for a maximum of 5 min and then check a 50-μL drop of sample under an inverted light microscope with a 4x objective.
      NOTE: Clusters of 10-15 cells are desirable, as this increases cell survival after electroporation.
   4. Transfer the cell suspension to a low-binding 15 mL tube and halt the dissociation by adding 9 mL of basal medium without antibiotics (see Table 2). Centrifuge at room temperature, 5 min at 600 x g, then discard the supernatant and resuspend the pellet in 1 mL of reduced serum medium (see Table of Materials).
   5. Count the number of cells with a Bürker's chamber and use a minimum of 1 x 10⁵ cells per electroporation reaction. Add 9 mL reduced serum medium to the 15 mL tube and centrifuge at room temperature, 3 min at 400 x g.
3. Electroporation
   NOTE: The following sections provide instructions on how to perform electroporation and to make organoids recover afterwards.
   1. Remove all of the supernatant and resuspend the pellet in an electroporation solution (see Table of Materials). Add a total amount of 10 μg DNA to the cell suspension and add electroporation solution to a final volume of 100 μL and keep the cell-DNA mixture on ice. Use CRISPR-concatamer vectors in combination with a Cas9 expression plasmid (e.g., Addgene #41815) in a 1:1 ratio.
      NOTE: The total volume of the DNA added should be less than or equal to 10% of the total reaction volume.
   2. Include a separate transfection mix containing a GFP plasmid to evaluate transfection efficiency (e.g., pCMV-GFP, Addgene #11153, or any generic GFP-expressing plasmid).
   3. Add the cell-DNA mixture to the electroporation cuvette and place it in the electroporator chamber. Measure the impedance by pushing the appropriate button on the electroporator and ensure that it is 0.030-0.055 kΩ. Perform electroporation according to the settings shown in Table 3.
      NOTE: If the impedance value falls outside of the allowed range, adjust the solution volume in the cuvette.
   4. Add 400 μL of electroporation buffer + Y-27632 to the cuvette and then transfer all to a 1.5 mL tube. Incubate at room temperature for 30 min to allow cells to recover and subsequently spin them at room temperature for 3 min at 400 x g.
   5. Remove the supernatant and resuspend the pellet in 20 μL/well of basement matrix. Seed approximately 1 x 104 to 1 x 105 cells per well in a 48-well plate and add EN + CHIR99021 + Y-27632 + 1.25% v/v DMSO medium. Incubate at 37 °C.
   6. On day 5, change the medium to EN + CHIR99021 + Y-27632, and check transfection efficiency by observing GFP expression (Figure 2). Keep organoids at 37 °C and refresh EN + CHIR99021 + Y-27632 medium after 2 days.
   7. On day 9, change the medium to WENR + Nic + Y-27632 and incubate at 37 °C.
      NOTE: Y-27632 can be removed after 7-10 days (on days 16-19).

	Cassette 1	Cassette 2	Cassette 3	Cassette 4
Sequence (5′-3′)	CACCGG[gRNA1]GT	ACCGG[gRNA2]G	CCGG[gRNA3]	ACACCGG[gRNA4]GTT
Sequence (5′-3′)	TAAAAC[RC-gRNA1]CC	AAAAC[RC-gRNA2]C	AAAC[RC-gRNA3]	CTAAAAC[RC-gRNA4]CCG

Table 1: Overhangs for Each Cassette of the CRISPR-concatemer Vector.

Basal medium		Comments
Store at 4 °C for 4 weeks
Cell culture medium	500 mL	See table of materials
L-Glutamine	100x 5 mL
Buffering agent 1 M	5 mL	See table of materials
Penicillin Streptomycin	100x 5 mL
WENR + Nic (Wnt + EGF + Noggin + Rspondin + Nicotinamide)
Store at 4 °C for 2 weeks
Basal medium	up to 50 mL
Neuronal cell serum-free supplement (50x)	1 mL	See table of materials
Neuronal cell serum-free supplement (100x)	500 μL	See table of materials
n-Acetylcysteine (500 mM)	125 μL
mouse EGF (100 μg/mL)	25 μL
mouse Noggin (100 μg/mL)	50 μL
R-Spondin conditioned medium	5 mL
Wnt3a conditioned medium	25 mL
Nicotinamide (1 M)	250 μL
EN + CHIR + Y-27632 (EGF + Noggin + CHIR + Y-27632)
Store at 4 °C for 2 weeks
Basal medium w/o Penicillin Streptomycin	up to 20 mL
Neuronal cell serum-free supplement (50x)	400 μL	See table of materials
Neuronal cell serum-free supplement (100x)	200 μL	See table of materials
n-Acetylcysteine (500 mM)	50 μL
mouse EGF (100 μg/mL)	10 μL
mouse Noggin (100 μg/mL)	20 μL
Y-27632 (10 μM)	20 μL
CHIR99021 (8 μM)	10 μL
EN (EGF + Noggin)
Store at 4 °C for 4 weeks
Basal medium	up to 50 mL
Neuronal cell serum-free supplement (50x)	1 mL	See Table of materials
Neuronal cell serum-free supplement (100x)	500 μL	See Table of materials
n-Acetylcysteine (500 mM)	125 μL
mouse EGF (100 μg/mL)	25 μL
mouse Noggin (100 μg/mL)	50 μL

Table 2: Organoid Media Composition.

	Poring pulse	Transfer pulse
Voltage	175V	20V
Pulse length	5 msec	50msec
Pulse interval	50msec	50msec
Number of pulses	2	5
Decay rate	10%	40%
Polarity	+	+/-

Table 3: Electroporation Settings.

Representative Results

Figure 1: Schematic Representation of the CRISPR-concatemer with 4 Cassettes. Scheme of the 4 gRNA-concatemer vectors with each 400 bp cassette containing a U6 promoter, two inverted repeated BbsI sites (also indicated as BB) and gRNA scaffold in this order. During the shuffling reaction, BbsI sites are replaced by gRNA fragments with matching overhangs and consequently lost. Binding sites of the sequencing primers for checking the correct insertion of gRNA oligos are shown by the blue arrows. Fwd = forward primer, Rev = reverse primer, Link 1/2/3 = linker regions 1/2/3. Please click here to view a larger version of this figure.

Figure 2: Representative Digestion Patterns of Concatemer Vectors.
(A) Double digestion of 3 and 4 gRNA-concatemer vectors with EcoRI and BglII. The correct digestion pattern is marked by a green tick, whereas vectors with only 1 or 2 gRNA insertions are marked by a red cross. Lane 1 shows digestion of a 4 gRNA-concatemer parental vector used as a positive control (marked by "+"); similarly, lane 5 shows digestion of a 3 gRNA-concatemer parental vector, marked by "+". (B) Digestion with BbsI, showing the correct size of undigested concatemer vectors (indicated by the green ticks). Digestion of a gRNA-containing concatemer vector that has lost BbsI sites is used as a positive control and is marked by "+".

Materials

Name	Company	Catalog Number	Comments
Optimized CRISPR Design Tool	Feng Zhang group		CRISPR gRNA design tool; http://crispr.mit.edu/
Webcutter 2.0			restriction mapping tool; http://rna.lundberg.gu.se/cutter2/
T4 PNK (Polynucleotide Kinase)	New England Biolabs	M0201L
T4 DNA ligase buffer	New England Biolabs	M0202S
T7 DNA Ligase	New England Biolabs	M0318L
DTT (dithiothreitol)	Promega	P1171
ATP (adenosine triphosphate)	New England Biolabs	P0756S
FastDigest BbsI (BpiI)	Thermo Fisher	FD1014
Tango buffer (BSA-containing restriction enzyme buffer)	Thermo Fisher	BY5
BglII	New England Biolabs	R0144
EcoRI	New England Biolabs	R0101
Plasmid-safe exonuclease	Cambio	E3101K
Thermal cycler	Applied biosystems	4359659
10G competent E. coli bacteria	Cambridge Bioscience	60108-1
Advanced DMEM/F12(cell culture medium)	Invitrogen	12634-034
Glutamax (L-Glutamine)	100x Invitrogen	35050-068
HEPES 1 M (buffering agent)	Invitrogen	15630-056
Penicillin-streptomycin 100x	Invitrogen	15140-122
B27 supplement (Neuronal cell serum-free supplement) 50x	Invitrogen	17504-044
N2 supplement (Neuronal cell serum-free supplement) 100x	Invitrogen	17502-048
n-Acetylcysteine 500 mM	Sigma-Aldrich	A9165-5G
Mouse EGF 500 μg/mL	Invitrogen Biosource	PMG8043
Mouse Noggin 100 μg/mL	Peprotech	250-38
Nicotinamide 1 M	Sigma	N0636
R-Spondin conditioned medium	n.a.	n.a.	Produced in house from HEK293 cells, for details see Sato and Clevers 2013
Wnt conditioned medium	n.a.	n.a.	Produced in house from HEK293 cells, for details see Sato and Clevers 2014
Y-27632 10 μM	Sigma-Aldrich	Y0503-1MG
Standard BD Matrigel matrix	BD Biosciences	356231
48-well Plate	Greiner Bio One	677980
CHIR99021	Sigma-Aldrich	A3734-1MG
IWP-2	Cell Guidance Systems	SM39-10
TrypLE (recombinant protease)	Invitrogen	12605-010
Opti-MEM (reduced serum medium)	Life technologies	51985-034
Electroporation Cuvettes 2 mm gap	NepaGene	EC-002S
Low binding 15 mL tubes	Sigma-Aldrich	CLS430791
Bürker’s chamber	Sigma-Aldrich	BR719520-1EA
NEPA21 Super Electroporator	NepaGene	contact supplier
Protein LoBind tubes low binding	Thermo Fisher	10708704
BTXpress electroporation buffer	Harvard Apparatus	45-0805
DMSO (Dimethyl sulfoxide)	AppliChem	A3672