This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.
CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.
The reverse genetics approach is a widely used method for investigating the function of a gene. In particular, loss-of-function studies, in which disruption of a gene causes phenotypic alterations, play a key role in building our understanding of biological processes. The CRISPR/Cas9 method represents the most recent advancement in genome engineering technology and has revolutionized the current practice of genetics in cells and organisms. Cas9 is an RNA-guided endonuclease which binds to a specific DNA sequence complementary to the gRNA and generates a double-strand break (DSB). This DSB recruits DNA repair machinery that, in the absence of a DNA template for homologous recombination, will re-ligate the cut DNA strand via error-prone non-homologous end joining, which can thus result in insertions or deletions of nucleotide(s) causing frameshift mutations1.
The great ease and versatility of the CRISPR/Cas9 approach has made it a highly attractive tool for genome-scale knockout screens aimed at unraveling unknown gene functions2,3. Nevertheless, single gene knockout approaches are of limited use if multiple paralogues with redundant functions exist. Thus, ablating a single gene might not be enough to determine the function of that gene given possible compensation by paralogues resulting in little or no phenotypic alteration4. It is therefore important to knock out paralogues in parallel by delivering multiple gRNA vectors targeting the different paralogous genes in order to overcome the influence of genetic compensation.
To extend the use of CRISPR/Cas9 to paralogous gene knockout, we have recently developed a rapid, one-step cloning method to clone up to four pre-annealed gRNAs into a single retroviral vector5. The backbone, named CRISPR-concatemer, is based on an MSCV retroviral plasmid containing repetitive gRNA expression cassettes. Each cassette contains two inverted recognition sites of the Type IIS restriction enzyme BbsI, which can be irreversibly replaced by an annealed gRNA oligo with matching overhangs using a Golden Gate shuffling reaction in a single tube6. This cloning method consists of repetitive cycles of digestion and ligation that allow simultaneous assembly of multiple DNA fragments by exploiting the different overhang sequences generated by BbsI. The uniqueness of this enzyme is, for instance, the ability to perform asymmetric cuts outside of its recognition sequence; therefore, each cassette can have a different sequence with customized overhangs flanking BbsI core site and in this way, each gRNA can be cloned in a specific position and orientation of the concatemer vector.
As a proof of principle, we demonstrated the use of this strategy in mouse intestinal organoids by disrupting simultaneously two pairs of paralogous negative regulators of the Wnt pathway by one round of electroporation5.
During the past few years, many other groups have developed similar strategies based on multiple gRNA expression vectors constructed using Golden Gate shuffling7 to achieve multi-gene knockout in various model systems, such as human cell lines8,9, zebrafish10 and Escherichia coli11. In their protocols, gRNAs are first cloned into individual intermediate vectors and then assembled together into one final product. By contrast, the main advantage of our CRISPR-concatemer strategy is the convenience of a single BbsI shuffling, cloning step. Like other gRNA concatemers, our method makes possible either the simultaneous knockout of up to four different genes or increased CRISPR knockout efficiency following the targeting of one or two genes with multiple gRNAs (Figure 1).
In this protocol, we describe in detail every step in the generation of CRISPR-concatemer vectors, from gRNA design to the Golden Gate reaction and to confirmation of successful cloning. We also provide a highly efficient protocol for the transfection of CRISPR-concatemers into mouse small intestinal organoids by electroporation and subsequent growth factor withdrawal experiments.
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.
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.
2. Cloning of gRNAs into the CRISPR-concatemer Vector
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 cultures14.
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.
4. Growth Factor Withdrawal
Note: Here it is exemplified how to conduct a growth factor withdrawal experiment when knocking out negative regulators of the Wnt pathway in intestinal organoids.
In order to confirm the presence of the correct number of gRNA inserts in the concatemer vector, restriction digestion is performed with enzymes (EcoRI + BglII) flanking all gRNA expressing cassettes (each cassette size is ~400 bp, Figure 1). For example, when generating a 4 gRNA- concatemer vector, the expected size of the lower band in the agarose gel is approximately 1.6 Kbp; any band lower than this indicates that not all of the 4 gRNA cassettes are inserted into the vector (Figure 2A). In addition, it is always recommended to check that all BbsI recognition sites are lost and the enzyme does not cut the vector (Figure 2B).
Once the constructs have been confirmed, they can be delivered to mouse intestinal organoids by electroporation to achieve optimal levels of transfection efficiency (up to 70%), as shown by the GFP control (Figure 3).
Finally, to functionally test the efficiency of this strategy, intestinal organoids transfected with Cas9 and concatemer vectors against Axin1/2 and Rnf43/Znrf3 were cultured in EN (R-spondin withdrawal) and EN + IWP2 (R-spondin and Wnt withdrawal, IWP2: Porcupine inhibitor, 2.5 μM) media for a minimum of 3 passages (Figure 4). While untransfected WT organoids died under both conditions, Axin1/2 knockout organoids survived in both due to downstream activation of the Wnt pathway; in addition, Rnf43/Znrf3 mutant organoids survive in the absence of R-spondin but cannot survive in the presence of IWP2, which causes depletion of the Wnt that activates the pathway. Taken together, these observations demonstrate that knockout of these pairs of paralogues is possible by generating the expected organoid phenotype. Details of these results have been published in Entwicklungsbiologie5.
Figure 1: Schematic Representation of the CRISPR-concatemer with 4 Cassettes. Scheme of the 4 gRNA-concatemer vector 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 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 "+". Please click here to view a larger version of this figure.
Figure 3: Representative Image of Successfully Electroporated Intestinal Organoids. Transfection of a GFP plasmid is instrumental to evaluate transfection efficiency. Approximately 24 h after electroporation, organoids containing a small number of cells are already visible and, if the electroporation procedure was successful, up to 70% of them displays green fluorescence. BF = bright field, GFP = green fluorescent protein. Scale bar = 2,000 µm. Please click here to view a larger version of this figure.
Figure 4: Representative Images of Mutant Intestinal Organoids. Knockout of the negative regulators of the Wnt pathway Axin1 and Rnf43, together with their paralogues, renders the intestinal organoids resistant to growth factor deprivation. In particular, Axin1/2 knockout organoids (Axin1/2 KO) can grow in the absence of both R-spondin (EN: EGF + Noggin) and Wnt (EN + IWP2: EN + Porcupine inhibitor), whereas Rnf43/Znrf3 mutant organoids (R&Z KO) can only survive in the absence of R-spondin (EN). In contrast, WT organoids can only survive in the control culture condition, WENR + Nic (Wnt + EGF + Noggin + R-spondin + Nicotinamide). Scale bars = 1,000 µm. Please click here to view a larger version of this figure.
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 + R-spondin + 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.
In this protocol, we detail all the steps necessary to generate CRISPR-concatemers and to apply CRISPR-concatemers in mouse intestinal organoids in order to simultaneously knock out multiple genes. As previously noted, this strategy has several advantages, such as its speed, high efficiency and cost-effectiveness.
In order to successfully perform the whole procedure, there are a few critical aspects to consider. First, it is essential that all gRNA oligos are properly annealed and phosphorylated, as they represent the starting material for the BbsI cloning reaction that in itself is very efficient. Secondly, when electroporating organoids, the more cells used per condition, the higher the maximum possible transfection efficiency. In addition, it is also important that after cell dissociation, small cell clusters predominate over single cells.
Nevertheless, it is possible to encounter technical problems when attempting either the cloning or the transfection for the first time; in the case of problems during gRNA cloning, it is recommended to double check the gRNA oligo sequence and, if correct, select additional bacterial colonies for restriction digestion screening. If transfection efficiency and cell viability are low post-electroporation, then it is advisable to repeat the protocol using more cells per condition and reducing the time of cell dissociation to 3 min.
Although the generation of CRISPR-concatemers is relatively cheap and easy, performing larger scale genetic screens in organoids is not, as the scale is limited by the costs associated with organoid culture and by its labor-intensive nature. It is worth mentioning in this case that the CRISPR-concatemer method is also compatible with cell lines, such as HEK293 and mouse embryonic stem cells.
Regardless of the cellular system, another potential drawback of this strategy can be encountered when aiming at the simultaneous knockout of three or four different genes. For instance, each gRNA will have a different targeting efficiency and the changes of hitting all the genes at the same time can be relatively low; for this reason, it is advisable to employ the concatemer system to direct more than one gRNA against the same gene.
Alternative strategies similarly based on Golden Gate shuffling have been proposed over the years to generate multiplex gRNA vectors7,8. However, in our method it is possible to directly assemble multiple gRNAs into a single retroviral vector in a single round of cloning, which makes it suitable for generating gRNA libraries to target paralogues.
Our CRISPR-concatemer is built in the MSCV retroviral vector backbone. Thus, gRNA concatemer-containing retrovirus can be used to generate stable cell lines that overexpress gRNAs. When combined with a Cas9-inducible system, one can perform inducible paralogue knockouts using our system.
In summary, here we describe how to clone up to four different gRNAs into the same vector in one step and how to apply this strategy to organoid culture with a high transfection efficiency. Furthermore, we provide useful suggestions to maximize the chances of success throughout the entire procedure.
The authors have nothing to disclose.
We thank Christopher Hindley for the critical reading of the manuscript. A.M. is supported by Wntsapp (Marie Curie ITN), A.A-R. is supported by the Medical Research Council (MRC), and B-K.K. and R.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and receive support through a core grant from the Wellcome Trust and MRC to the Wellcome Trust – MRC Cambridge Stem Cell Institute.
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 | |
Plasmid mini kit | Qiagen | 12125 | |
Table top microcentrifuge | Eppendorf | UY-02580-01 | |
Inoculating loops | Microspec | PLS5 | |
Bacteria incubator | Sanyo | MIR-262 | |
Luria-Bertani broth (LB) | Sigma-Aldrich | L3522 | |
Agarose | Sigma-Aldrich | A4718 | |
Agarose gel electrophoresis apparatus | Bioneer | A-7020 | |
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 2013 |
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 2mm 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 |