Articles in PresS. Am J Physiol Renal Physiol (December 4, 2014). doi:10.1152/ajprenal.00612.2014

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Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing

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Ankita Roy1, Joshua H. Goodman1, Gulnaz Begum2, Bridget F. Donnelly1, Gabrielle Pittman1,

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Edward J. Weinman4, Dandan Sun2,5, and Arohan R. Subramanya1,3,5

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Departments of 1Medicine, 2Neurology, and 3Cell Biology, University of Pittsburgh School of

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Medicine, Pittsburgh, Pennsylvania, USA; 4Department of Medicine, University of Maryland

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Medical School, Baltimore, Maryland; 5VA Pittsburgh Healthcare System, Pittsburgh

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Pennsylvania, USA.

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Article Type: Innovative Methodology

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Running Title: CRISPR/Cas9 edited WNK1 knockout cells

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Contact:

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Arohan R. Subramanya, MD

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Department of Medicine, Renal-Electrolyte Division

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University of Pittsburgh School of Medicine

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S828A Scaife Hall

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3550 Terrace Street

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Pittsburgh, PA 15261

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Tel 412.624.3669 Fax: 412.647.6222

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Email: [email protected]

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Copyright © 2014 by the American Physiological Society.

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Abstract

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Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and

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chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These

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cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases

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through their downstream effector kinases, SPAK and OSR1. Multiple WNK kinases can coexist

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within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt

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transport remain incompletely understood. Deletion of specific WNKs from cells that natively

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express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here,

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we outline a simple method to selectively knock out full length WNK1 expression from

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mammalian cells using RNA-guided CRISPR/Cas9 endonucleases. Two clonal cell lines were

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generated by using a single guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which

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produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced

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endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability.

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Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased

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expression of the KLHL3/Cullin-3 E3 ubiquitin ligase complex, and was rescued by exogenous

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WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable

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from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory

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volume increase (RVI) following hypertonic stress, confirming an essential role for WNK1 in cell

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volume regulation. Collectively, our data show how this new, powerful, and accessible gene

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editing technology can be used to dissect and analyze WNK signaling networks.

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Keywords: WNK1, SLC12 cotransporters, Genome editing, CRISPR/Cas system

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Introduction

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SLC12 cotransporters mediate the electroneutral movement of cations with Cl- across membranes

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(10). To date, nine SLC12 family members have been identified. Among these is a subgroup of

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sodium dependent cation chloride cotransporters, which include the bumetanide-sensitive Na+-

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K+-2Cl- cotransporters NKCC1 (SLC12A2) and NKCC2 (SLC12A1), and the thiazide-sensitive

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Na-Cl cotransporter, NCC (SLC12A3). In the kidney, NKCC2 and NCC are expressed at the

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apical membrane of epithelial cells of the thick ascending limb of the loop of Henle (TAL) and

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the distal convoluted tubule (DCT), respectively. Both of these cotransporters reabsorb sodium

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with chloride from the tubular lumen, and thus help control extracellular fluid volume and blood

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pressure (10). In contrast, NKCC1 is nearly ubiquitously expressed and plays an important role in

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the maintenance of cell volume and intracellular chloride concentration (24). NKCC1 is also

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enriched in the basolateral membranes of the renal collecting duct and polarized secretory

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epithelia, where it facilitates the vectorial movement of ions into the tubular lumen (11, 22, 24,

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46).

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In order for the sodium-dependent SLC12 cotransporters to be maximally active, they must be

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phosphorylated at specific serines and threonines harbored within their intracellular N-termini

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(31). A network of serine threonine kinases mediates this process. Direct phosphorylation of the

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cotransporters is carried out by two homologous tonicity responsive serine threonine kinases,

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Ste20- and SPS1-related proline alanine rich kinase (SPAK) and oxidative stress responsive

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kinase 1 (OSR1) (27, 44). Immediately upstream of SPAK and OSR1 are the With-No-Lysine

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(WNK) kinases, which activate SPAK and OSR1 via T-loop phosphorylation (48). Mammals

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harbor four WNK paralogs; three of these, WNK1, WNK3, and WNK4, are expressed in the

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kidney (25, 33). All of the WNKs share a similar domain architecture, consisting of an N-terminal

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serine-threonine kinase domain, an autoinhibitory domain (AID), and several coiled-coil and

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SPAK/OSR1 binding domains (31). The coiled coil domains facilitate the formation of WNK

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homo- and heterooligomers (4, 21, 42). Once associated in complexes, the kinase domains can

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trans-autophosphorylate their partners, while the AIDs can cross-inhibit the activity of other

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associated WNKs (42, 51, 52). Thus, WNK kinases assemble into higher order regulatory

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complexes to carry out their downstream effects on SPAK/OSR1 activity and electroneutral salt

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transport. The stability of these complexes is likely dependent on their oligomeric composition

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and stoichiometry, and through interactions with the Kelch-like 3/Cullin 3 (KLHL3/CUL3) E3

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ubiquitin ligase complex (29, 45).

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A broad range of hormones and osmotic stressors that stimulate sodium dependent cation chloride

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cotransport utilize the WNK-SPAK/OSR1 cascade to mediate their effects (31, 38). Some

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evidence suggests that these diverse physiological stimuli convergently trigger SPAK/OSR1

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phosphorylation by signaling through specific WNKs (3, 41). However, it remains unclear

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whether a single WNK kinase, or whether the coordinated action of multiple WNKs are required

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to carry out a specific physiological response. Tackling this question in whole animal models is

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challenging due to a number of issues, including the heterogeneity of cell types in renal

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parenchyma, the discrepancies in WNK and SPAK/OSR1 expression in different segments of the

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nephron, and the embryonic lethality of deleting some components of the network, including

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WNK1 and OSR1 (49, 54). A commonly used alternative strategy is to knock down endogenous

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WNKs in cell lines that express an intact WNK-SPAK/OSR1 network by RNA interference

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(RNAi). A drawback of this approach, however, is that siRNA-mediated gene silencing is rarely

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100% effective at eliminating the expression of a target gene product. Indeed, in our own studies,

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we have only been able to reduce the expression of a specific WNK kinase to about half of its

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native abundance (39); similar results have been reported by others (4). Thus, RNAi experiments

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can only support, but not verify, whether a specific WNK kinase is essential for transducing a

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specific physiological response. To resolve this critical question, cell lines that completely lack

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the expression of one or more WNKs are needed.

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To determine the role of the individual WNKs in signal transduction and cation chloride

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cotransporter activation, we are using CRISPR RNA-guided Cas9 nucleases to knock out

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components of the WNK-SPAK/OSR1 signaling pathway in mammalian cells. In the present

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study, we describe how we used this novel method to generate a mammalian WNK1 knock out

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cell line. In our experience, the technique is straightforward, cost-effective, and efficient,

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permitting the generation of multiple genetically and biochemically validated knockout clones

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within weeks. We report that WNK1 knockout cells exhibit a substantial reduction in WNK4

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expression and SPAK/OSR1 activity. As expected, they also display impaired volume regulation

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in response to hypertonic stress, consistent with a critical role for WNK1 in mediating NKCC1-

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dependent regulatory volume increase (RVI). We also report that total WNK1 deletion increases

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the abundance of the KLHL3/CUL3 complex, suggesting a new layer of feedback regulation

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between WNK1 and its cognate E3 ubiquitin ligase.

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Materials and Methods

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Antibodies, Plasmids, and Reagents

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We used the following antibodies in this study: rabbit polyclonal anti-N terminal WNK1 (exon 1;

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Sigma), rabbit polyclonal C-terminal WNK1 (exon 28; Sigma), rabbit polyclonal anti-WNK4

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(Novus Biologicals (39)), rabbit polyclonal anti-WNK2 (ab28852; Abcam), rabbit polyclonal

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anti-SPAK (C-terminal; Cell Signaling Technology (34)), rabbit polyclonal anti-OSR1 (#3729;

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Cell Signaling); rabbit polyclonal anti-Cullin3 (ab1871; Abcam (1)), rabbit polyclonal anti-

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Kelch-like 3 (ab66655; Abcam (2, 26)), mouse monoclonal anti-Actin (Sigma); rabbit polyclonal

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anti-Tubulin, (Bethyl). The rabbit polyclonal anti-WNK3 antibody was manufactured by

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Millipore (Cat #07-2262) in collaboration with the University of Dundee’s Division of Signal

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Transduction Therapy. The immunogen was a glutathione-S-transferase (GST) fusion protein to

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residues 1142-1461 of the human protein. The immunogenicity of this recombinant protein was

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confirmed in previous studies (42). The rabbit polyclonal anti-phospho S373/S325 SPAK/OSR1

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(“S motif”) antibody, validated by immunoblotting in lysates of hypotonic low Cl--stimulated

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HEK-293 cells treated with and without lambda phosphatase was from Millipore (Cat #07-2273).

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Secondary HRP-conjugated antibodies raised in goat were from Jackson ImmunoResearch. The

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bicistronic pX330 vector containing cDNAs encoding human codon-optimized S. pyogenes Cas9

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(hSpCas9) and an adaptable CRISPR RNA (crRNA)/ trans-activating crRNA chimera containing

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adjacent BbsI cloning sites for protospacer guide sequence insertion was purchased from

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Addgene (plasmid #42230). To generate the N-terminal HA-tagged L-WNK1-pcDNA3.1

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construct, a 5’ EcoRI-PacI L-WNK1 fragment encoding the HA tag was swapped with the

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corresponding 5’ end of the original N-terminal myc-tagged L-WNK1 cDNA (50) in pcDNA3.1,

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using standard subcloning methods. All reagents were purchased from Sigma unless otherwise

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noted.

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WNK1 single guide RNA (sgRNA) expression vector construction

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A 20bp guide sequence (5’–GCACTCTGCGGGACAGCCGC–3’) targeting DNA within the first

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exon of WNK1 was selected from a published database of predicted high-specificity protospacer–

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PAM

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CACCGCACTCTGCGGGACAGCCGC–3’ and 5’–AAACGCGGCTGTCCCGCAGAGTGC–

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3’) containing the WNK1 guide sequence and BbsI ligation adapters were synthesized by IDT.

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100μM of each oligo was annealed using T4 polynucleotide kinase (New England Biolabs, NEB)

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and 1µL 10X T4 Ligation Buffer in a total volume of 10μl in a BioRad thermal cycler. The

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cycling conditions were: 370C for 30min, then 950C for 5 min, followed by a ramp to 250C at

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50C/min. The annealed oligo was ligated into the Bbs1-digested pX330 vector using 5 µL of 2X

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QuickLigation Buffer, 1 µL of QuickLigase (New England Biolabs, NEB). The ligation mixture

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was treated with PlasmidSafe exonuclease (Epicentre) and transformed in OneShot® chemically

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competent Stbl3 cells (Life Technologies). Following plasmid DNA extraction (Qiagen), the

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sequence of the construct was verified by automated DNA sequence analysis performed at the

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University of Pittsburgh Genomics and Proteomics Core Laboratories (GPCL).

target

sites

in

the

human

exome

(23).

Two

complementary

oligos

(5’–

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DNA mismatch-specific (T7E1) endonuclease assay

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HEK293T cells in 6 well dishes were cultured as describe previously to 50-60% confluence (28).

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Cells were transfected with 1μg of WNK1 sgRNA plasmid and 5μl of Lipofectamine 2000 per

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well. Cells were also transfected with pX330 empty vector as a control. 72h post transfection, the

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cells were harvested and genomic DNA was extracted (QuickExtract DNA extraction solution,

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Epicentre). A region of exon 1 of the WNK1 gene was amplified with genomic DNA specific

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primers (forward primer, 5’–CCCTTCACGCCCTTTTCGTTCACGAATCC–3’; reverse primer,

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5’–ACCTGCAGTTCACACCAGGCGACTTCC–3’). Homoduplex PCR products were

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denatured and rehybridized using stepdown annealing conditions to generate homo and

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heteroduplexes. The mixture of duplexes was treated with T7E1 nuclease for 15 min at 370C

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(NEB). The reaction was stopped using 1.5μl of 0.25M EDTA, and the products were analyzed

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on a 2% agarose gel.

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Transfection and cell sorting

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HEK293T cells were cultured in 6-well dishes to 70-80% confluence. Cells were cotransfected

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with 1μg of WNK1 sgRNA plasmid plus 1μg of pEGFP-N1 (Clontech) and 5μl of Lipofectamine

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2000 per well. pEGFP-N1 derived enhanced green fluorescent protein (eGFP) was used as a

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fluorescent marker to sort transfected cells. 48h post transfection, cells were pelleted in PBS+ 2%

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FBS and sorted in 96 well plates using a FACSAris II cell sorter (BD BioSciences). Single cells

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from two populations of GFP-expressing cells (high expression and medium expression) were

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expanded to obtain individual clones.

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Clone validation

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Individual clones were lysed in detergent solution for 20 min at 40C as described previously (8).

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The lysate was centrifuged at 16,000g for 10 min and 20μg of supernatant was fractionated on 4-

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20% SDS PAGE gels, transferred to nitrocellulose, and screened by immunoblotting with WNK1

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antibodies. Genomic DNA was isolated from edited clones and non-edited HEK293T control

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cells as described above. Exon 1 of WNK1 was PCR amplified using the WNK1-specific PCR

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primers described above. The PCR products were A-tailed and cloned into pGEM-T Easy

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(Promega). Individually cloned amplicons were then analyzed by Sanger sequencing (GPCL). For

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imaging studies evaluating cellular morphology, cells were plated on Biocoat coverslips (BD),

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fixed for 30 min in 2% paraformaldehyde and evaluated by differential interference contrast

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(DIC) microscopy using a Leica DM 6000 epifluorescence/ DIC microscope equipped with a

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Retiga 400R digital imaging camera.

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Reverse Transcriptase PCR (RT-PCR)

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To detect the mRNA expression of endogenous WNK kinases in HEK-293T cells, RNA was

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extracted from unedited cells using Trizol (Life Technologies), and the RNA was reverse

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transcribed to cDNA using the iScript cDNA synthesis kit (Biorad). RT-PCR reactions for the

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four WNK kinases were carried out using the following primer sets: WNK1-Forward: 5’–

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CGTCTGGAACACTTAAAACGTATCT–3’;

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CACCAGCTTCTTAGAACTTTGATCT–3’

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ACGTCTATGCCTTTGGGATGT–3’; WNK2-Reverse: 5’–GATCTCGTACCTTTCCTCCTT

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GT–3’ (14); WNK3-Forward: 5’–ATTCAAGATAGCCCTGCACAAT–3’; WNK3-Reverse: 5’–

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GTCAGAGGAATGGATCAGAAG–3’

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TGCCTTGTCTATTCCACGGTCTG–3’;

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CAGCTGCAATTTCTTCTGGGCTG –3’ (18).

WNK1-Reverse: (43);

(12);

WNK2-Forward:

WNK4-Forward: WNK4-Reverse:

5’– 5’–

5’– 5’–

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Cell volume regulation studies Cell volume change was determined using calcein as a marker of intracellular water

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volume, as established previously (20). Briefly, cells on coverslips were incubated with 0.5 µM

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calcein-AM for 30 min at 37°C. The cells were placed in a heated (37°C) imaging chamber

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(Warner Instruments, Hamden, CT) on a Nikon Ti Eclipse inverted epifluorescence microscope

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equipped with perfect focus, a 40X Super Fluor oil immersion objective lens, and a Princeton

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Instruments MicroMax CCD camera. Calcein fluorescence was monitored using a FITC filter set

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(excitation 480 nm, emission 535 nm, Chroma Technology, Rockingham, VT). Images were

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collected every 60 sec with MetaFluor image-acquisition software (Molecular Devices,

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Sunnyvale, CA) and regions of interest (~20-30 cells) were selected. Baseline drift resulting from

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photobleaching and dye leakage was corrected as described before (20). The fluorescence change

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was plotted as a function of the reciprocal of the relative osmotic pressure and the resulting

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calibration curve applied to all subsequent experiments as described before (20). The HEPES-

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buffered isotonic solution contained (in mM, pH 7.4): 100 NaCl, 5.4 KCl, 1.3 CaCl2, 0.8 MgSO4,

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20 HEPES, 5.5glucose, 0.4 NaHC03. Anisosmotic solutions (250, 317 and 800 mOsm) were

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prepared by removal or addition of Sorbitol to the above solution. Osmolarity was determined

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using an osmometer (Advanced Instruments, Norwood, MA).

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Results

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Generation of WNK1 knockout cell lines

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Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are RNA

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programmable host defense mechanisms in bacteria and archaea that degrade foreign nucleic acid

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(15). In 2013, multiple groups engineered the type II CRISPR system of Streptococcus pyogenes

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to edit genes in mammalian cells (6, 17, 23). This was accomplished by optimizing the S.

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pyogenes Cas9 enzyme for expression and localization in eukaryotic nuclei, and by fusing the

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essential noncoding RNA components of the CRISPR locus into a single guide RNA (sgRNA)

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whose 5’ end could be programmed with a 20bp “guide sequence” for gene-specific targeting.

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While the 3’ end of the sgRNA associates with the Cas9 enzyme, the 5’ guide sequence will bind

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to its complementary genomic target through Watson-Crick base pairing. The target is always

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positioned next to a “protospacer adjacent motif” (PAM). In S. pyogenes, the PAM corresponds

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to the sequence “NGG”, where N can be any nucleotide. When bound to this target site, the

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sgRNA-associated Cas9 enzyme generates a double stranded break near the PAM that undergoes

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imperfect repair by non-homologous end joining (NHEJ). This results in the formation of

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insertions or deletions (“indels”) that create a frameshift mutation and premature termination of

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protein synthesis (30).

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We first set out to identify an appropriate target site in WNK1 for gene editing. The site was

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selected from a bioinformatic database of 23bp target-PAM sequences in the human exome that

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was filtered to minimize off-target crossreactivity (23). We focused on exon 1 of WNK1 as a

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putative target site, because NHEJ-mediated frameshifts within this region could potentially

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terminate translation upstream of the N-terminal kinase domain, thereby eliminating kinase

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activity (Fig. 1A). From a list of several candidates, we chose a target sequence spanning bases

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325-344 of the human WNK1 cDNA (CCDS8506.1). On the minus strand of the WNK1 gene,

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these nucleotides are positioned immediately 5’ to the trinucleotide PAM sequence “GGG” (Fig.

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1B). Once identified, an oligo pair containing the guide sequence was cloned into pX330, a

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bicistronic vector that contains the Cas9 enzyme and the programmable chimeric sgRNA (6). For

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our initial attempt at generating a WNK knockout cell line, we elected to use HEK293T cells as

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the model system. We chose this cell line for two reasons. First, HEK239T cells are commonly

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used to edit genes with CRISPR/Cas technology, making them a well-established model to test

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the efficacy of RNA-guided endonucleases in deleting WNK kinase expression. Second,

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HEK293T cells are frequently used model to study cation chloride cotransporter activity,

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trafficking, and regulation by the WNK-SPAK/OSR1 pathway.

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To test the effectiveness of the WNK1 sgRNA at triggering Cas9-mediated gene editing at the

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target site, HEK293T cells were transiently transfected with the pX330-WNK1sgRNA plasmid,

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resulting in the generation of a mixed population of edited and non-edited cells. 72h post

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transfection, we extracted this heterogeneous pool of genomic DNA, amplified a genomic region

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containing the target site by PCR, denatured and reannealed the PCR amplicons in a thermal

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cycler to generate heteroduplex pairs, and subjected these rehybridized products to digestion with

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T7E1 nuclease. The T7E1 enzyme selectively recognizes and cleaves mismatched heteroduplexes

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harboring indels (5). Because CRISPR/Cas9 complexes trigger DSBs and imperfect NHEJ near

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the PAM (6), we predicted that T7E1 digestion of mismatches in the target site should generate

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DNA fragments of approximately 384-437 bp in size (Fig. 2A, right). Indeed, we found that in

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genomic DNA extracts from WNK1 sgRNA-transfected cells, the T7E1 enzyme cleaved exon 1

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of WNK1 at a position targeted by the WNK1 sgRNA, resulting in the generation of lower

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molecular weight DNA fragments of about 400bp (Fig. 2A, left).

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Because the T7E1 screening assay suggested that the WNK1 sgRNA was functional, we next

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sought to generate clonal WNK1 knockout cell lines. We cotransfected a new batch of HEK293T

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cells with the px330-WNK1sgRNA and a plasmid encoding eGFP, and isolated transfected cells

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by FACS. The sorted cells were then plated as individual clones in 96 well plates and expanded.

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18 clones were probed for WNK1 protein expression by western blotting. From this initial screen,

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two clones (clones 4 and 5) were selected for further analysis. Both clones showed a complete

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absence of WNK1 expression compared to the unedited control, when probed with antibodies

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directed to the extreme N- and C-terminal ends of the protein (Fig. 2B). Despite the complete

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absence of WNK1 protein expression, differential interference contrast (DIC) microscopy

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indicated that the cellular morphology of the two clones was grossly indistinguishable from wild-

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type controls (Fig. 2C).

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To characterize the nature of gene editing in these cells, we PCR amplified exon 1 from genomic

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DNA extracted from the two individual clonal populations and analyzed the products on an

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agarose gel. We noted a substantial shift in the migration of the PCR products of both the

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knockout clones compared to the control DNA (Fig. 2D), indicating that the CRISPR/Cas9

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machinery introduced deletions into the genomic sequence. To further confirm these findings, the

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PCR products were TA cloned and 12 clonal amplicons from each knockout cell line were

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sequenced. In clone 4, all 12 sequences exhibited a 105bp deletion, which encompassed the target

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site + PAM (“mutant allele 1”; Fig. 2E and Table 1). This would be predicted to cause a

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frameshift mutation that generates a 91 amino acid truncated protein lacking kinase activity

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(p.Arg81Hisfs*10 per Human Genome Variation Society (HGVS) nomenclature (7); Table 1).

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Sequence analysis of amplicons from clone 5 revealed a mixed population of mutated alleles. In 7

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of the 12 amplicons, we detected the same 105bp deletion that was present in clone 4. The other 5

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amplicons revealed a second 34bp deletion allele that also encompassed the target site + PAM

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(“mutant allele 2”; Fig. 2E and Table 1). The predicted consequence of this deletion is a

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frameshift mutation that generates 5 new amino acids and a premature stop codon downstream of

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alanine 109, also resulting in a kinase dead fragment (pAla109Thrfs*5; Table 1). Thus, in both

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clonal cell lines, we were unable to detect intact wild type alleles, and all of the genomic DNA

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sequences that were identified contained deletions that created nonfunctional gene products.

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Taken together with the complete lack of detectable WNK1 protein expression by western

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blotting with multiple WNK1-specific antibodies, these findings confirm the generation of two

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WNK1 knockout cell lines using the CRISPR/Cas9 system.

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Deletion of WNK1 decreases WNK4 but not WNK3 expression

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Several studies suggest that WNK kinases assemble and interact in macromolecular complexes

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(21, 40, 42, 52). Thus, we set out to determine how the deletion of WNK1 protein expression in

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cells affects the abundance of the other WNK kinases and associated proteins in the pathway. We

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first performed RT-PCR studies to identify native WNK mRNA transcripts in wild type

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HEK293T cells. These assays confirmed the expression of WNK1, WNK3, and WNK4 (Fig. 3A),

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but we were also able to detect WNK2, a brain-expressed kinase (33). When we probed wild type

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and WNK1 knockout cells for WNK-SPAK/OSR1 protein expression, we found that WNK1

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deletion strongly reduced the steady state total abundance of WNK4 by approximately 65 percent

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(Fig. 3B). WNK2 expression was also significantly reduced, although to a lesser degree. In

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contrast to WNK4 and WNK2, we observed an increase in WNK3 abundance. The levels of

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active phosphorylated SPAK and OSR1, detected with an antibody which recognizes

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phosphorylation of the C-terminal regulatory domain common to both kinases (34), were much

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lower in the knockouts compared to the wild-type, though the levels of total SPAK and OSR1 did

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not vary significantly. Collectively, these data confirm previous observations that WNK1 is a

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major determinant of SPAK and OSR1 activation status. The effects of WNK1 on downstream

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SPAK/OSR1 activity, however, may be due in part to its cooperative effects on the stability and

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activation status of other WNK kinases, such as WNK4.

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A major potential pitfall with the CRISPR/Cas9 system concerns the relatively short 20bp length

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of the sgRNA guide sequence. Although Cas9-mediated gene editing only occurs at target sites

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localized immediately adjacent to a PAM (16), the short length of the guide increases the risk of

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cross-reactivity with target sites in different genes that share a similar sequence (19). Such off-

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target effects may lead to the misinterpretation of experimental results. To rule out the possibility

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that the reduced abundance of WNK4 observed in WNK1 knockout cells was due to an off-target

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effect, we performed a “rescue” experiment with heterologously expressed WNK1. For these

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studies, clone 4 WNK1 knockout cells were transiently transfected with full-length kinase active

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WNK1, and 48h post transfection we quantified WNK4 protein abundance by immunoblotting.

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Consistent with an on-target effect, reconstitution of WNK1 protein expression significantly

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increased WNK4 protein abundance (Fig. 4). These findings indicate that the reduced WNK4

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abundance seen in WNK1 knockout cells is due to a WNK1-specific effect on WNK4 protein

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expression.

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The E3 ubiquitin ligase CUL3 and its adaptor protein KLHL3 function in a protein complex that

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interacts with and degrades WNK1 and WNK4 (29, 45). A recent study provided evidence that

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the interaction between KLHL3-CUL3 and WNK4 can be regulated physiologically (36). We

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therefore wondered if the absence of WNK1 protein expression upregulates the abundance of

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KLHL3-CUL3, providing a potential explanation for the reduction in WNK4 protein abundance.

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Consistent with this hypothesis, we observed a two- to threefold increase in the steady state

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protein expression of KLHL3 and CUL3 in WNK1 knockout cells (Fig. 3B). Although additional

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studies will be needed to further evaluate the significance of this finding, the data suggest that

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WNK1 can regulate the stability of the KLHL3-CUL3 complex through feedback, and that the

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abundance and activity of WNK4 may be influenced by this regulation.

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WNK1 deletion reduces SPAK/OSR1 activation and impairs regulatory volume increase (RVI)

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Hyperosmotic stress activates WNK1 by multisite phosphorylation (50, 53). To test the effect of

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hypertonicity on WNK1-dependent activation of SPAK/OSR1, we monitored the abundance and

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phosphorylation status of several components of the network after subjecting wild type and

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WNK1 knockout cells to a hypertonic solution containing sorbitol. We elected to treat cells with

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0.5M sorbitol (800mOsm) for 30 minutes, a previously reported hypertonic stimulus that activates

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the WNK-SPAK/OSR1 pathway (53). Consistent with previous observations, exposure of wild-

349

type cells to 800mOsm sorbitol solution reduced the electrophoretic mobility of WNK1 (Fig. 5A),

350

a phenomenon that has been attributed to enhanced WNK1 phosphorylation and activation (53).

351

Moreover, hypertonic stress did not significantly reverse the dramatic inhibitory effect of WNK1

352

deletion on WNK4 expression. In contrast, although WNK2 protein abundance was lower in

353

WNK1 knockout cells under unstressed isotonic conditions, its protein abundance recovered

354

dramatically during hypertonic stress, such that its expression was indistinguishable from WNK2

355

in sorbitol-treated wild type cells (Figs. 5A, B). The abundance of WNK3 was also significantly

356

increased by either WNK1 knockout (Figs. 5A, B, and 3B; compare WNK3 signal in WT and KO

357

cells under isotonic conditions) or hypertonicity.

358

We also evaluated the effect of hypertonic stress on downstream SPAK/OSR1 expression and

359

activity. Unexpectedly, and despite their structural homology, sorbitol treatment had different

360

effects on total SPAK and OSR1 protein abundance. In wild-type cells, OSR1 expression was

361

dramatically increased by sorbitol hypertonicity, while SPAK expression did not change

362

significantly (Figs. 5A, B). The net result of these effects, however, was a significant increase in

363

net SPAK/OSR1 phosphorylation status. WNK1 knockout cells exhibited similar trends in SPAK

364

and OSR1 total protein abundance. Thus, WNK1 knockout cells exhibited an overall increase in

365

SPAK/OSR1 phosphorylation in response to hypertonicity; however, relative to wild type cells,

366

this phosphorylation was 50% reduced (Fig. 5A, B; compare pSPAK/pOSR1 signal under

367

hypertonic conditions in WT and WNK1 KO cells).

368

Previous studies have suggested that WNK1 plays an important role in the cellular response to

369

hypertonic stress. Following hypertonicity-induced shrinkage, cells recover their volume by

370

stimulating NKCC1-mediated Na-K-2Cl cotransport, and by inhibiting potassium and chloride

371

efflux via K-Cl cotransporters (KCCs) (13). This process, termed regulatory volume increase

372

(RVI), is directly linked to cotransporter phosphorylation status (13). WNK1 is believed to be an

373

important mediator of this process, since it triggers SPAK/OSR1-mediated stimulatory

374

phosphorylation of NKCC1 while simultaneously influencing the phosphorylation of KCCs at

375

key inhibitory residues that block its activity (9, 27, 32, 44). To definitively test this hypothesis,

376

we used a previously validated cell volume measurement assay with the fluorescent dye calcein

377

(20) to determine whether deletion of WNK1 affects RVI in HEK293T cells during osmotic

378

challenge. WNK1 wild type and knockout cells were exposed to a 20-minute hypertonic stress

379

with 0.5M sorbitol. WNK1 WT cells exhibited an immediate sharp drop in cell volume, followed

380

by a slow volume increase (10.7±3%/min), representing RVI (Fig. 5C, slope 1). At the end of the

381

20-min hypertonic stress, WNK1 WT cells recovered to 64% of the initial volume (63.5 ± 7.0). In

382

contrast, WNK1 KO cells underwent more severe cell shrinkage and failed to show any RVI (Fig.

383

5C), with a recovery rate of -1.6 ± 0.3%/min. Both WNK1 WT and KO cells recovered their

384

original volume upon returning to the isosmotic solution (Fig. 5C, slope 2). Inhibiting NKCC1

385

activity with bumetanide blocked the RVI response in WNK1 WT cells (Fig. 5D). However,

386

bumetanide did not exhibit any effects in WNK1 KO cells (Fig. 5E). Taken together, these data

387

further suggest that WNK1 deletion weakens the RVI response, which is in part likely due to

388

impaired SPAK/OSR1-NKCC1 activation.

389

Discussion

390

In this study, we used CRISPR/Cas9 technology to knock out WNK1 gene expression in

391

HEK293T cells, a model commonly used to study WNK-SPAK/OSR1 regulation of cation

392

chloride cotransport. Our findings indicate that an sgRNA programmed to a target site in exon 1

393

of the WNK1 gene triggers sequence specific Cas9-mediated NHEJ, resulting in the total ablation

394

of WNK1 protein expression. Using two separate clones, we found that WNK4 expression is

395

dependent on the presence of WNK1. In addition, WNK1 deletion attenuated the normal RVI

396

response to hypertonicity, implicating WNK1 as an essential regulator of cell volume. The

397

methodology presented here offers a simple and broadly applicable framework for generating

398

validated knockout cell lines. Our workflow is adapted from previously published methods (30),

399

and can be divided into four phases: (1) guide sequence design and sgRNA construction, (2)

400

sgRNA screening, and (3) clone isolation, and (4) clone screening and validation. In our

401

experience, this straightforward approach can be used to generate gene-edited clones within a

402

matter of weeks. The technique also provides certain distinct advantages, since it permits the

403

study of endogenous genes that are resistant to conventional RNAi, or are embryonically lethal

404

when knocked out in vivo. In theory, the approach could be applied to any cell culture model that

405

can tolerate FACS and can receive cDNA via transfection or viral transduction. Thus, the gene

406

editing workflow presented here could potentially be adapted to a variety immortalized renal cell

407

types, including tubular epithelial cells, podocytes, pericytes, or other cells relevant to kidney

408

physiology.

409

RNA programmable gene editing is a rapidly evolving technology with a growing number of

410

applications (15). Though we used the technique to knock out a single gene in this study,

411

CRISPR/Cas9 nucleases can also be harnessed for multiplex genome engineering (6, 23). Thus,

412

sgRNAs targeting distinct sites within the genome can be coexpressed to knock out multiple

413

genes simultaneously. This approach can potentially be used to rapidly eliminate redundancy in

414

signaling pathways. For example, the multiplexing approach could be applied to the WNK-

415

SPAK/OSR1 pathway to genetically delete several or all of the WNK kinases, or perhaps both

416

SPAK and OSR1. This may help define the physiological roles of specific network components,

417

or identify new kinases that phosphorylate cation chloride cotransporters independently of

418

SPAK/OSR1. This multiplexing technique has been applied to the manipulation of genes in

419

cultured cells, and has also been used to knock out multiple genes from mice (47). Equally

420

intriguing, CRISPR/Cas systems can also knock-in point mutations or new sequence into genes.

421

In this case, a donor DNA template containing the desired sequence modification is introduced

422

into cells with the Cas9 enzyme and sgRNA, triggering gene editing through homology-directed

423

repair (HDR) (23). Other than the requisite introduction of this donor template, the workflow for

424

generating mutant clones is analogous to that outlined in this study.

425

The expanding armamentarium of CRISPR/Cas-related applications is quickly pushing it to the

426

forefront of currently available tools for in vitro and in vivo gene modification. However, the

427

system has important caveats that should be considered. The first and most significant of these is

428

the potential for CRISPR to trigger off-target gene disruption (19). The short 20nt guide sequence

429

that directs Cas9 to a given target site may cross-react with other sequences present in the genome.

430

It is therefore critically important to design guide sequences with high target specificity. Off-

431

target cross-reactivity occurs at sites that pair with the 3’-most 13bp of the guide sequence,

432

termed the “seed sequence” (16, 23). In this study, we chose a WNK1 guide sequence that binds

433

perfectly to a single 23bp target site (including the NGG PAM) in the human exome, but does not

434

pair with unwanted target sites that harbor a matching seed sequence (23). Although this in silico

435

approach helps minimize off target cleavage, databases such as the one used in our study are still

436

in the process of being optimized (23). Moreover, single base pair mismatches within the seed

437

sequence may not completely abrogate off-target binding (16), and in silico reference sequences

438

may not be a perfect match to the genome of an immortalized cell line. Thus, it is essential to

439

carry out supplemental experiments verifying that the ablation of a target gene of interest is

440

responsible for a specific physiological effect. In our study, we used two approaches to test the

441

specificity of WNK1 deletion on WNK4 abundance. First, we surveyed the WNK-SPAK/OSR1

442

pathway in two genetically distinct knockout cell lines that completely lack WNK1 protein

443

expression, and confirmed that all of the known major components of the signaling pathway were

444

similarly affected. During this analysis, we observed that both clones exhibited increased

445

expression of the KLHL3/CUL3 E3 ligase complex, suggesting a potential mechanism that might

446

decrease WNK4 protein abundance. Second, we performed a “rescue” experiment in which

447

exogenous WNK1 was transiently transfected into knockout cells and WNK4 expression was

448

compared to vector transfected knockouts and wild type controls. These studies confirmed that

449

reconstituting WNK1 back into the network restored WNK4 abundance. Although we did not

450

pursue other validation studies, an alternative approach would be to evaluate additional WNK1

451

knockout clones generated through the usage of a second sgRNA targeting a different area of the

452

WNK1 gene. Because this nonoverlapping sgRNA should generate a different complement of off-

453

target events, similar experimental results would be more likely to be the consequence of target

454

gene knockout. Finally, deep sequencing of knockout cell lines would be a comprehensive gold

455

standard to guide study interpretation. Though such studies can be costly, it seems advisable to

456

pursue such characterization if multiple genes are going to be knocked out, or if edited cells will

457

be used to generate large “unbiased” mRNA or proteomic datasets.

458

Another important issue that should be considered is the possibility that endogenous protein

459

expression may drift during cell passage. In our hands, we found that the endogenous expression

460

of certain components of the WNK-SPAK/OSR1 signaling pathway in HEK cells, particularly

461

WNK3, can change over multiple cell passages. More specifically, the increased WNK3

462

expression noted in the knockouts (Figs 3 and 5) was lost over multiple passages, and the WNK3

463

protein levels drifted down to that of WT cells (data not shown). Thus, comparisons between

464

knockout and control cells with significant discrepancies in passage number may not necessarily

465

be reflective of target gene ablation. For this reason, we recommend analyzing control and

466

knockout cell lines within a tight passage window and performing side-by-side comparisons with

467

multiple genotypically distinct knockout clones.

468

The studies presented here highlight the importance of the WNK-SPAK/OSR1 pathway in cell

469

volume regulation. We found that in wild type cells, sorbitol-induced hypertonic stress shifted the

470

electrophoretic mobility of WNK1. Previous work indicates that this alteration in mobility

471

correlates with enhanced phosphorylation and activation, suggesting a role for WNK1 in

472

SPAK/OSR1 activation by hypertonicity (53). Consistent with this, WNK1 knockout cells

473

exposed to sorbitol hypertonicity exhibited reduced SPAK/OSR1 regulatory domain

474

phosphorylation and a blunted RVI response. These results demonstrate that WNK1 plays a

475

critically important role in cell volume regulation, either acting by itself, or via co-regulation with

476

WNK4, whose expression was strongly downregulated in WNK1 knockout cells. Despite the

477

absence of significant WNK1 and WNK4 expression during hypertonicity, SPAK/OSR1

478

phosphorylation was not completely abolished. This suggests that other kinases could

479

phosphorylate SPAK and OSR1 upon osmotic stress. WNK3 and WNK2 were detectable during

480

sorbitol hypertonicity; thus, these other WNKs could mediate this process. Alternatively, there

481

are WNK-independent mechanisms that can selectively activate OSR1, whose total protein

482

abundance was strongly increased by hypertonicity. One such activation mechanism could be via

483

the recently described P13K-mTORC2-OSR1 pathway (35).

484

The WNK1 knockout cell lines have also started to provide new information on the behavior of

485

endogenously expressed WNK kinases in cells expressing a functional WNK-SPAK/OSR1

486

network. Our data indicate that WNK1 influences the total protein abundance of other WNK

487

kinases. Specifically, WNK1 knockout cells exhibited a strong decrease in the abundance of

488

WNK4, and to a lesser degree WNK2. This could be due to changes in the composition of WNK

489

complexes, as previous studies have suggested that the WNKs can hetero-oligomerize via their C-

490

terminal coiled coil domains (42). Alternatively, WNK1 might regulate proteins that destabilize

491

the other WNK kinases. Indeed, we found that WNK1 deletion increases the abundance of the

492

KLHL3/CUL3 complex, a known negative regulator of WNK4 abundance (37). This suggests

493

that WNK1 may control WNK4 ubiquitylation and degradation through feedback inhibition of

494

KLHL3/CUL3. Though the mechanism by which WNK1 deletion downregulates WNK4

495

abundance will require further study, our data clearly show that selectively knocking out one

496

WNK kinase can dramatically influence the abundance of others. This has important implications

497

for the correct interpretation of knockdown and heterologous overexpression studies that

498

implicate a specific WNK kinase in a particular physiological response. Most of these types of

499

studies have not evaluated the effects of WNK overexpression on other endogenous components

500

of the pathway. Yet, for most of the studies conducted in heterologous expression systems such as

501

Xenopus oocytes or HEK293 cells, this unmonitored endogenous network is co-opted for at least

502

some of the intermediate signaling stream that triggers downstream cation chloride cotransporter

503

activation. In order to fully ascertain the role of specific WNKs in signal transduction, it seems

504

that future studies would benefit from the development of gene edited cell lines that contain

505

solitary WNK kinases, or a “WNKless” cell line in which specific WNK-dependent signaling

506

cascades can be reconstituted by rescue.

507

In conclusion, we report a simple approach that uses programmable CRISPR/Cas9 nucleases to

508

generate knockout cell lines. The new WNK1 knockout model generated via this methodology

509

highlights new relationships between WNK1, WNK4, and other associated proteins, and confirms

510

an essential role for WNK1 in NKCC1-dependent cell volume recovery.

511

demonstrate how this new and powerful genome-editing tool can be used to dissect and analyze

512

WNK signaling networks.

513

Thus, our data

514 515

Acknowledgments

516

The study was supported by National Institute of Health Grants R01DK098145 (to A.R.S.),

517

P30DK79307 (Pittsburgh Center for Kidney Research), R01 NS038118 and NS075995 (to D.S.),

518

R01DK55881 (to E.J.W.), a Mid-Level Career Development Award from the US Department of

519

Veteran’s Affairs (to A.R.S.), and funds from the U.S. Department of Veterans Affairs Middleton

520

Award (to E.J.W.). We thank Erich Wilkerson for FACS sorting, and Deborah Steplock and

521

Alexandra Socovich for excellent technical assistance. We are grateful to Drs. John Aach

522

(Department of Genetics, Harvard Medical School, Boston, MA), Mike Butterworth, Adam

523

Kwiatkowski, and Anatalia Labilloy for helpful discussions.

524 525

Disclosures

526

None

527

528 529

References

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Figure Legends

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Figure 1. WNK1 sgRNA and target site. (A) Top, Domain structure of full-length kinase active

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human WNK1 (also referred to as “L-WNK1” (40)). The location of the kinase domain, three

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coiled coil domains (CC), proline rich domains, an autoinhibitory domain (AID), and C-terminal

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SPAK/OSR1 binding motifs are shown. Bottom, Schematic representation of the WNK1 gene.

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The sgRNA target site is located at the 5’ end of exon 1, which encodes amino acids upstream of

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the kinase domain activation loop. (B) Close-up representation of the sgRNA target site. The

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20nt guide sequence comprising the 5’ end of the chimeric sgRNA is shown. This sequence pairs

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with the DNA target site (indicated on the bottom strand). Immediately 3’ to the target sequence

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is the trinucleotide PAM (5’-NGG). The Cas9 enzyme introduces a DSB near the PAM,

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triggering imperfect NHEJ.

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Figure 2. Generation and validation of WNK1 KO cell lines. (A) Mismatch-specific

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endonuclease assay. Genomic PCR (gPCR) products spanning exon 1 of WNK1 were amplified

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from the template of a heterogeneous population of HEK293T cells transfected with px330-

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WNK1 guide. Rehybridization and treatment with T7E1 nuclease results in fallout bands of ~380-

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430 bp, shown by a bracket. This is consistent with the predicted cleavage sizes of 384 and 437bp.

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This fallout signal was not observed in gPCR amplicons from cells transfected with an empty

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px330 plasmid lacking the WNK1 guide. (B) Cell lysates (20μg) of wild type and clonal WNK1

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knockout isolates (“Clone 4” and “Clone 5”), probed with exon 1 and exon 28 specific WNK1

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antibodies. A parallel set of lysates (20µg) was probed with anti-actin antibody as a loading

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control. (C) DIC images of wild type control, and WNK1 knockout clones. High magnification

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images of areas bounded by the white boxes are shown below the corresponding low power

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image. (D) Following clone isolation, gPCR amplicons encompassing the modified locus of

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genomic DNA were run on a 1% agarose gel. The products from both WNK1 knockout clones

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migrate faster than the unedited control. (E) Sequence alignment of bases 5736-5885 of exon 1 of

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the RefSeqGene human WNK1 sequence (NG_007984.2), a wild type allele from control

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HEK293T cells, and two mutant alleles identified from each of the two WNK1 knockout clones

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by genomic PCR. “Mutant Allele 1” was identified in both clones 4 and 5; “Mutant Allele 2” was

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only isolated from clone 5. The complementary plus strand sequences corresponding to the 20nt

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target and 3nt PAM are highlighted in dark gray and black, respectively. See also Table 1.

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Figure 3. Steady state expression of the endogenous WNK-SPAK/OSR1 pathway in wild

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type and WNK1 knockout cells under isotonic conditions. (A) Agarose gel electrophoresis of

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RT-PCR products from wild type HEK-293T cells (“WT”) and template-free negative controls

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“neg”. Predicted PCR amplicon sizes are provided to the right of each image. (B) Steady state

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protein abundance of WNK-SPAK/OSR1 pathway components in wild type (“WT”) and WNK1

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knockouts (“KO” Clones 4 and 5). Total protein abundance of WNK2 and WNK4 were decreased

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in the WNK1 knockout cell lines, while WNK3 expression was increased. SPAK/OSR1

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activation was decreased in the WNK1 knockout cells. The abundance of CUL3 and KLHL3

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were similarly increased in WNK1 KOs. n=4; •p