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A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines

Background: Multiple myeloma is the second most common hematologic neoplasm which is characterized by proliferation and inltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to treatment resistance. The CRISPR/Cas9 gene editing system has been recently used in several cell lines and mouse models to understand the role of various genetic alterations in pathogenesis of hematologic malignancies. Despite the recent advances of gene edition tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy. Results: We have designed different guide RNAs and cloning them into a lentiviral plasmid, which contain all the machinery necessary for develop the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids for the production of lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Conclusions: We have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines.


Background
Multiple myeloma (MM) is a hematologic neoplasm of plasmatic cells that in ltrates the bone marrow and secrete monoclonal immunoglobulins. MM is the second most common hematologic malignancy in the world, and despite the therapeutic advances, the disease is associated with a poor outcome, due to disease relapsing and the development of treatment resistances (1)(2)(3).
MM is preceded by a pre-malignant and asymptomatic stage called monoclonal gammopathy of undetermined signi cance (MGUS) and intermediate stage termed smoldering multiple myeloma (SMM).
Then, most patients develop the symptomatic stage known as MM and characterized by the monoclonal protein secretion and the end-organ damage (1,2). Finally, MM cells can develop the ability of proliferation outside the bone marrow and produce more aggressive stages such as extramedullary multiple myeloma (EMM) or plasma cell leukemia (PCL) (1,2).
How the asymptomatic MGUS stage become a MM remains unknown, but many factors are involved, being the genetic alterations the most frequent causes of disease progression. Also, cells present in the bone marrow microenvironment play an important role in MM progression. These cells, that include bone marrow stromal cells, endothelial cells, and other hematopoietic cells, produce chemokines and other factors that interact with MM cells favoring their migration to the bone marrow and facilitating their proliferation and survival (4).
Rho GTPases are small GTP-binding proteins with an important role in converting extracellular signals into a large range of cellular responses, including cell adhesion, cell-cycle progression, cell migration, cell morphogenesis, gene expression, and actin cytoskeleton dynamics (5)(6)(7). Therefore, Rho proteins have been widely described as important regulators of tumour cell proliferation, survival, and invasion (8). Rho proteins also play an important role in the development of hematological neoplasms since they are involved in chemotaxis and motility in lymphoid lines through ROCK-LIMK pathway (9). Speci cally, RhoU, an atypical GTPase Rho protein, has been recently related to MM progression, as its expression increases in MGUS patients, and it is downregulated as the disease progresses (10). Rnd3 protein is also an atypical GTPase that belongs to Rho family and it has been involved in cancer progression and drug resistance in different type of cancers (11). However, the possible role of Rnd3 in hematological neoplasm remains unknown.
The CRISPR/Cas9 gene editing system has been recently used in several cell lines and mouse models to understand the role of various genetic alterations in pathogenesis of hematologic malignancies, as well as discovering new therapeutic targets for future clinical stages (12,13). The CRISPR system is a bacterial adaptive immune system that requires the endonuclease Cas9 from Streptococcus pyogenes (or analogous proteins from other species) and a single guide of RNA (sgRNA). That guide leads the nuclease activity to complementary sequences in the substrate DNA, usually on the coding region (14). CRISPR/Cas9 is used for genome editing by introducing deletions on the protein coding ORF (open reading frame) using homology repair (HR) or nonhomologous end joining (NHEJ) repair. These deletions can lead to frame shifts that result in a loss of function of the encoded protein (15). CRISPR/Cas9 is a permanent technique that allows the generation of knockout cell lines by genome editing, but it has variable tolerance for mismatches between its sgRNA and the target DNA sequence, so off-targets effects are common with this technology (16). Nowadays, there are variants of this technique which need a catalytically inactivated Cas9, known as a dead Cas9 (dCas9). This dCas9 can be used to activate or repress gene expression when it is associated to a sgRNA directed to speci c gene promoter region. If the dCas9 is fused to a transcriptional activator such as VP64, the gene expression is activated, and it is known as CRISPR activation. However, dCas9 can be fused to a transcriptional repressor, most known as Krüppel-associated box (KRAB), that can induce DNA methylation and decrease the accessibility of chromatin at the enhancer and promoter regions and, therefore, represses gene expression at transcriptional level. This technology is called CRISPR interference (CRISPRi) (17,18). CRISPRi is frequently used to perform genetic screens in mammalian cells (18) and also is used to cell engineering and regenerative medicine like retinal, muscle, nerve, or bone degeneration among others (17).
Furthermore, CRISPRi can be used to silencing gene expression in speci c human cell types, such as neurons or iPSCs (19,20). This silencing can be constitutive or inducible, but frequently it is stable when it is achieved by lentiviral transduction.
In this work, we describe and analyze the e cacy of CRISPRi technology when it is used to silencing Rnd3 expression in MM cell lines by lentiviral transduction.

sgRNAs cloning into lentiviral plasmid pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro
To obtain the sequence of the sgRNA an Addgene library (CRISPRi library was a gift from Jonathan Weissman (Addgene #62217)) was consulted (21). From the sequences available directed to RND3 promoter, two different sgRNA were selected. In addition, another sgRNA guide to a non-coding region (scramble) was chosen to be used as negative control. To allow the cloning with the selected sgRNAs, a prior modi cation must be performed. In the forward and reverse primers, a nucleotide tails, target of Esp3I restriction enzyme must be introduced (table 1). Table 1 List of primers used to generate the sgRNAs. The necessary tails in the ends for the cloning were highlighted. The annealing of the primers designed was necessary to prepare a double stranded sgRNAs. This process requires the use of forward and reverse oligonucleotides, and their incubation at 95ºC, allowing a progressive cooling. Finally, T4 PNK enzyme was added to phosphorylate the 5' end for subsequent cloning ligation. The details of the process are described in Fig 1A. Then, the annealed sgRNAs were diluted 1:1000 and cloned into lentiviral plasmid construction pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro (this plasmid was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236; RRID:Addgene_71236) (22). 5µg of plasmid was linearized with Esp3I enzyme (Thermo Fisher) and then it was dephosphorylated with alkaline phosphatase (Promega) ( Fig   1B). The digested plasmid was checked using an agarose gel electrophoresis and the 15kb expected band was puri ed using NZYGelpure kit (Nzytech), following the manufacturer's instructions and quanti ed by Nanodrop Simplinano (GE Healthcare Life Science). The ligation reaction containing both the linearized plasmid and the annealed sgRNA was prepared and incubated overnight as described in Fig 1C. Finally, the ligation product was transformed into DH5α competent E. coli and grown on to LB-Agar plates with ampicillin. Bacterial colonies were double checked to demonstrate the ligation. Two different strategies can be used using plasmid DNA obtained by GenElute Plasmid Miniprep kit (Sigma-Aldrich): the ampli cation using a PCR with the speci c primers, and alternatively, by DNA sequencing (Fig 1D). Once checked, bacteria culture was ampli ed to obtain enough plasmid DNA using PureLink Plasmid Filter Midiprep kit (Invitrogen) following the manufacturer's instructions. These plasmids containing the sgRNAs cloned into the lentiviral vector were then used to be transfected in HEK 293T cells.
Transfection, production, and concentration of lentiviral particles The production of lentiviral particles was carried out by the calcium phosphate transfection method. HEK 293T lentiviral packaging cells were cotransfected with different vectors that included the sgRNA plasmids and 3rd generation lentiviral envelope and packaging plasmids ( Table 2). 24 hours prior the transfection, HEK 293T cells were seeded in 10cm plates and reached 60-70% of con uence the day of transfection. Two hours before transfection the medium was removed and replaced with 9 mL of fresh medium. To obtain a high virus titer, ve 10 cm plates per each sgRNA construction were needed although the information related to the volumes and quantities hereby is referred to one 10 cm culture plate. hours the transfection medium was removed, 6 ml of fresh medium was added, and cells were kept for 48 hours more. Then, to concentrate the lentiviral particles, the medium from the ve plates was recollected and centrifuged for 10 minutes at 2000g to discard remaining cells and the supernatant was ltered with 0.45 µm lter. To obtain high virus titer, the ltered supernatant was ultracentrifuged to 27100 rpm for 150 minutes at 4ºC and the pellet was resuspended in 1 ml of PBS for 30-60 minutes on ice, with occasional mix. The lentiviral particles were used to freshly transduce the MM cells or freeze at -80ºC to further infections.

Transduction of MM cells and Rnd3 knock-down (KD) generation
RPMI 8226 and JJN3 cells were transduced with concentrate lentiviral particles in 6-well plates. 100 µL of lentiviral particles and 1 µL of polybrene solution (10 mg/mL, Sigma-Aldrich) were added to 1x10 6 MM cells/well in 1ml nal volume and cells were kept at 37ºC and 5% CO 2 for 48 hours. Then, the transduction medium was removed, and MM cells were maintained in fresh medium. The selection of transduced cells was made by addition of puromycin (2µg/mL, Sigma-Aldrich) to the culture medium for 24 hours. Finally, puromycin resistant cells were ampli ed and Rnd3 silencing was checked by qPCR and western blot.
CRISPRi technology allows a gene silencing at transcriptional level. The sgRNAs lead the dCas9-KRAB to the gene promoter and inhibit its expression as described above. Figure 2A shows how transduced cells presented a signi cant decrease in RND3 mRNA expression. Likewise, Rnd3 silencing was demonstrated at protein level by western blot ( Figure 2B).

Discussion
Gene edition is a powerful tool for basic and clinical settings in cellular therapies for lymphoid malignancies. Considering different techniques for gene silencing, RNAis have been used in multiple studies with mammalian cells. Speci cally, this strategy has been used by many authors to downregulate the expression of speci c genes in MM cell lines (10,(23)(24)(25)(26)(27). RNAi technology acts at translational level because it facilitates the mRNA degradation therefore it produces a transient gene silencing. Despite of the advantages that this technique can provide it is described that could generate off-targets (28) that reduces the speci city of the gene repression. Therefore, other strategies to repress gene expression in MM cell lines have been tested such as CRISPR/Cas9. CRISPR/Cas9 technique allows the edition of the genome using the Cas9 endonuclease and sgRNAs to introduce deletions on the ORF. Nowadays, there are many studies that use CRISPR/Cas9 technology to repress gene expression in human cell lines and provides stable knockout cells for the study of pathologies progression (29). Despite the real advantages of the CRISPR/Cas9 genome edition, the production of off-target effects is common (16). Therefore, other strategies that reduce these off-target effects have been proposed such as high-delity Cas9 for genome editing or dCas9 for gene silencing.
CRISPR interference (CRISPRi) technology consists in using a sgRNA to direct a dCas9 fused to KRAB repressor to the gene promoter and downregulate gene expression at transcriptional level (18). Recently, many studies have been developed to inactivate the gene expression in human cells, like neurons or iPSCs (19,20).
Although the recent advances of gene edition tools, the use of CRISPR/Cas9 technology for gene editing of MM has not so far been extended. A recent study shows the generation of FAM46C knockout MM cell lines using CRISPR/Cas9 (30). In this work, we propose an extensive protocol for the using of CRISPRi technology to downregulate Rnd3 expression in RPMI 8226 and JJN3 MM cell lines, by using lentiviral vectors and speci c RND3 sgRNA. Our results demonstrated that transduced cells present a permanent reduced Rnd3 expression both at mRNA and protein level, thus allowing to generate stable knock-down MM cells and we propose the use of CRISPRi tool to downregulate gene expression of cell lines, such as MM cells, with a previous low success rate obtained with other molecular techniques.

Conclusions
We developed and validated an effective CRISPRi strategy using lentiviral transduction for gene silencing in multiple myeloma cell lines. This strategy combines the design of the sgRNAs, their cloning into the lentiviral vector with CRISPRi machinery, the transfection of HEK 293T cells for the production of the lentiviral particles and the transduction of MM cell lines with these particles for gene silencing. We demonstrated that this silencing is permanent and works both at mRNA and protein level, so this CRISPRi strategy allows the generation of stable knockdown cell lines.   Schematic protocol of sgRNA cloning into lentiviral plasmid. First, the annealing of the primers was required to generate the sgRNAs (A). Then, the lentiviral plasmid was digested with Esp3I enzyme to linearize, purify, and quantify (B). The ligation of the sgRNA into the linearized lentiviral plasmid was made at 16ºC overnight in presence of T4 ligase enzyme (C).Finally, the ligated plasmids were transformed in DH5α competent E. coli (D) and colonies were checked by PCR and sequencing, before DNA ampli cation.