A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

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.


Introduction
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 mutations 1 .
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 functions 2,3 . 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 alteration 4 . 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 vector 5 . 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 tube 6 . 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 electroporation .
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.

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   3. Exonuclease treatment NOTE: This step is highly recommended as it increases the efficiency of the cloning by removing any traces of linearized DNA. 1. Treat the BbsI shuffling reaction with a DNA exonuclease (see Table of

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 15 . Table 2 for all media compositions.

Please refer to
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% CO 2 in a humidified incubator (as previously described 15 ). 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.

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.  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  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 Ω. 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 10 4 to 1 x 10 5 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.

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.
1. 10-14 days after electroporation, split the organoids in a 1:3 ratio in a 48-well plate following the above-mentioned steps (3.2.1 -3.2.2). 2. Resuspend the organoid pellet in 20 µL of basement membrane matrix and let it solidify at 37 °C for 10 min. Then, overlay 250 µL of growth factor-deprived medium (e.g. EN) to test whether knockout of the target genes has been achieved 5 . 3. Split the organoids under growth factor-deprived conditions for a minimum of 2 -3 passages to see a difference in survival between wild type wildtype (WT) control organoids and mutant organoids 5,15 . NOTE: Wildtype organoids should not be able to survive in growth factor-deprived medium over two passages, while mutant lines should be able to grow.

Representative Results
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 gRNAconcatemer 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 Rspondin 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 Developmental Biology 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 vectors 7,8 . 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.

Disclosures
The authors have nothing to disclose. The authors have no conflict of interest declared.