Activation of the Anti-Aging and Cognition-Enhancing Gene Klotho by CRISPR-dCas9 Transcriptional Effector Complex
Multiple lines of evidence show that the anti-aging and cognition-enhancing protein Klotho fosters neuronal survival, increases the anti-oxidative stress defense, and promotes remyelination of demyelinated axons. Thus, upregulation of the Klotho gene can potentially alleviate the symptoms and/or prevent the progression of age-associated neurodegenerative diseases such as Alzheimer’s disease and demyelinating diseases such as multiple sclerosis. Here we used a CRISPR-dCas9 complex to investigate single-guide RNA (sgRNA) targeting the Klotho promoter region for efficient transcriptional activation of the Klotho gene. We tested the sgRNAs within the − 1 to − 300 bp of the Klotho promoter region and identified two sgRNAs that can effectively enhance Klotho gene transcription. We examined the transcriptional activation of the Klotho gene using three different systems: a Firefly luciferase (FLuc) and NanoLuc luciferase (NLuc) coincidence reporter system, a NLuc knock-in in Klotho 3′-UTR using CRISPR genomic editing, and two human cell lines: neuronal SY5Y cells and kidney HK-2 cells that express Klotho endogenously. The two sgRNAs enhanced Klotho expression at both the gene and protein levels. Our results show the feasibility of gene therapy for targeting Klotho using CRISPR technology. Enhancing Klotho levels has a therapeutic potential for increasing cognition and treating age-associated neurodegenerative, demyelinating and other diseases, such as chronic kidney disease and cancer.
KeywordsAlzheimer’s disease Multiple sclerosis Neuroprotection Myelin Chronic kidney disease Cancer
clustered regularly interspaced short palindromic repeat
polymerase chain reaction
Dulbecco’s modified Eagle’s Medium
phosphate buffered saline
fetal bovine serum
bovine serum albumin
sodium dodecyl sulfate polyacrylamide gel electrophoresis
The anti-aging protein Klotho was named after the goddess who spins the thread of life (Kuro-o et al. 1997). Klotho knockout mice have an accelerated aging phenotype recapitulating many of the features observed in aged humans (Kuro-o et al. 1997). Conversely, lifespan was extended by ~ 30% and an increased resistance to oxidative stress was observed in Klotho overexpressing mice (Kurosu et al. 2005). The single copy gene Klotho is a type I transmembrane protein which is mainly expressed in the brain, kidney, and reproductive organs (Masuda et al. 2005). Klotho is also shed by proteolytic cleaving resulting in a soluble form that is detectable in serum and CSF (Bloch et al. 2009; Chen et al. 2007; Matsumura et al. 1998). A third form of Klotho, found mainly in the brain, results from differential mRNA splicing and is secreted from the cell into the blood and CSF (Massó et al. 2015). Both the transmembrane and soluble forms of Klotho have pleiotropic actions throughout the body and are essential for many homeostatic functions (for extensive reviews see (Abraham et al. 2016; Kuro-o 2012). Our lab demonstrated that Klotho protects neurons from oxidative stress by increasing expression of antioxidant factors, and promotes oligodendrocyte maturation in vitro. Furthermore, we showed that Klotho induces remyelination in vivo in the cuprizone-induced demyelination model of MS (Zeldich et al. 2015). We further reported that Klotho overexpression reduces cognitive deficits in a mouse model of Alzheimer’s disease, and that Klotho enhances cognition in humans and mice (Dubal et al. 2014; Dubal et al. 2015). Together, these findings suggest that increasing Klotho levels in the brain would have a beneficial effect to prevent cognitive impairment associated with normal aging and neurodegenerative diseases.
Recent advances in genome editing technology may provide a means to increase Klotho expression using CRISPR (clustered regularly interspaced short palindromic repeat) technology. CRISPR technology utilizes RNA guide sequences to target a specific gene or genomic locus where CRISPR-associated (Cas) proteins are utilized for genomic editing (Cho et al. 2013; Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013). Single or multiple loci can be targeted simultaneously by the simple base-pair complementarities between an engineered single-guide RNA (sgRNA) and a target genomic DNA sequence. A nuclease-deficient Cas9 (dCas9) and the VP64 fusion transcription activator in complex with guide RNA (gRNA) is used to specifically activate single or multiple genes simultaneously (Gersbach and Perez-Pinera 2014; Kearns et al. 2014; Konermann et al. 2015). A recent study demonstrated that structure-guided engineering of a CRISPR-dCas9 complex mediated efficient transcriptional activation at endogenous genomic loci (Konermann et al. 2015). This result is based on the incorporation of bacteriophage coat protein MS2 RNA aptamers into the stem loop and loop two of the single-guided RNA (sgRNA2.0) that are exposed in the ternary Cas9 complex. The complex consists of three components: a dCas9-VP64 fusion, a MS2-P65-HSF1 activation helper protein, and a sgRNA. The incorporation of three distinct activation domains—VP64, P65, and HSF1—into the complex aids robust transcriptional activation through synergy (Konermann et al. 2015).
In this study, we report the application of the CRISPR-dCas9 complex to activate human Klotho gene expression. We found that two sgRNAs enhanced Klotho expression in two different cell reporter systems, the neuronal cell line SY5Y and the kidney cell line HK-2. These two cell lines express Klotho endogenously, but to different extents. The findings provide support for Klotho gene activation and demonstrate a potential therapeutic strategy for Klotho upregulation which may mitigate the symptoms of some neurodegenerative diseases and demyelinating disorders, and other diseases such as cancer and kidney disease.
Materials and Methods
Design and Cloning of sgRNA Plasmid
sgRNA1 sense: 5′-CACCGGGCATAAAGGGGCGCGGCGC-3′
sgRNA1 anti-sense: 5′-AAACGCGCCGCGCCCCTTTATGCCC-3′
sgRNA2 sense: 5′- CACCGCGGCGGGGCGCGGGCATAAA-3′
sgRNA2 anti-sense: 5′-AAACTTTATGCCCGCGCCCCGCCGC-3′
sgRNA3 sense: 5′-CACCGGTGCCTTTCTCCGACGTCCG-3′
sgRNA3 anti-sense: 5′-AAACCGGACGTCGGAGAAAGGCACC-3′
sgRNA4 sense: 5′-CACCGGAAACGTCCTGCACGGCTCC-3′
sgRNA4 anti-sense: 5′-AAACGGAGCCGTGCAGGACGTTTCC-3′
Cell lines were maintained under standard growth conditions and propagated in DMEM (Dulbecco’s modified Eagle’s medium) (4.5 g/ml glucose) containing 10% FBS (fetal bovine serum) (Atlanta Biologicals) and 1% penicillin/streptomycin (100 units/ml). For HK-2 kidney cells, DMEM:F12 (1:1) medium was used. All cell culture solutions were obtained from Cellgro unless otherwise noted in the text.
Cloning of Klotho (KL) 4 kb Promoter into FLuc and NLuc Luciferase Coincidence Reporter and Stable Cells Generation
5′-AAATCGATAAGGATCCGATGGAGCGGAGAATGGGCGG-3′ (forward primer)
5′-ATACGCAAACGGATCCGCTGTGGAATGTGTGTCAG-3′ (reverse primer)
The PCR product was isolated and ligated with pNL vector by digestion with BamHI to generate the pNLCoI1-SV40 vector. The KL 1.8 kb promoter was digested from pGL3-KL1800 (King et al. 2012) with HindIII and XhoI and subcloned into pNLCoI1-SV40 vector using the same restriction sites to generate pNLCoI1-SV40-KL1800.
5′-CTCGCTAGCCTCGAGATCTATAGTGCCACATGGTGAC (forward primer for 2.2 kb KL promoter insert)
(reverse primer for 2.2 kb KL promoter insert)
5′-GGGAAATGTGATACTCCATGTAG-3′ (forward primer for vector)
5′-CTCGAGGCTAGCGAGCTCAGGTACC-3′ (reverse primer for vector)
The insert and vector bands (100 ng each) were ligated together using In Fusion kit (Clontech) to generate pNLCoI1-SV40-KL4000. Stable HEK-293 cells expressing 4 kb of the KL promoter or the control PGK promoter with the coincidence reporter were generated from single clones and selected using Hygromycin (Invivogen, USA) at a concentration of 75 μg/ml for 2 weeks following Hygromycin 25 μg/ml for maintenance.
Generation of a NLuc Knock-in HEK293 Cell Line by CRISPR Genomic Editing
gRNA pair 1 sense: 5′-CACCGGTCTCACTGGCATCTTGTTG-3′
gRNA pair 1 anti-sense: 5′-AAACCAACAAGATGCCAGTGAGACC-3′
gRNA pair 2 sense: 5′-CACCGCAGGGACACAGGGTTTAGAC-3′
gRNA pair 2 anti-sense: 5′-AAACGTCTAAACCCTGTGTCCCTGC-3′
pcDNA3.1 reverse: 5′-AAGCTTCGTATATCTGGCCCGTACATCGCG-3′
KL 2710 forward: 5′-AGATATACGAAGCTTCCCACATACTGGATGGTATCAATC-3′
KL 3700 reverse: 5′-AGTAGCTCCGCTTCCGACAGGACCTCAAAAATCATATAA-3′
P2A NLuc forward: 5′-GGAAGCGGAGCTACTAACTTCAGCC-3′
P2A NLuc reverse: 5′-TTAGACGTTGATGCGAGCTGAAGC-3′
KL 3743 forward: 5′-CGCATCAACGTCTAATTGAGGGCCTTGCACATAGGAAAC-3′
KL 4978 reverse: 5′-CCCTCTAGACTCGAGATTATGAAAGAAGGCAAAAAGTTGC-3′
Klotho intron 4 forward: 5′- GTGTTGTGTGCAAAATACGTAATAA-3′
NLuc reverse: 5′- TGACATGGATGTCGATCTTCAG-3′
The forward primer is located in intron 4 of Klotho gene upstream to the left homology arm, and can therefore avoid false positive stable colonies with random insertion into genomic DNA. As a control for a specific activation of the Klotho promoter in a coincidence reporter vector, we have used pNLCoI4[luc2-P2A-NlucP/PGK/Hygro] Vector (cat. 1492, Promega, USA). For validation of the dual luciferase system, we used as positive controls Ataluren (PTC124) (cat. S6003, SelleckChem, USA) for FLuc and Cilnidipine (cat. S1293, SelleckChem, USA) for NLuc luciferase.
Cells were grown on poly-D-lysine-coated plates in 96-, 12-, or 6-well formats. Twenty-four hours after plating, cells reached 70–80% confluency and were transfected with a 1:1:1 ratio of Klotho specific targeting sgRNA plasmid or control sgRNA cloning backbone plasmid, MS2-P65-HSF1effector plasmid (Addgene, #61423), and dCas9-VP64 effector plasmid (Addgene, #61422). For positive controls, cells were transfected with a 1:2 ratio of Egr1 (a transcription factor known to activate Klotho transcription) or pcDNA3.1 empty vector. Transfections were carried out using Mirus TransIT-X2 with 100 ng, 1 μg, or 2 μg of total plasmid DNA per well in 96-, 12-, or 6-well plates, respectively. Transfection medium was removed and replaced with fresh medium after 5 h.
For measurement of FLuc and NLuc expression the coincidence reporter vector under Klotho promoter, Nano-Glo® Dual-Luciferase® Reporter Assay System (cat. N1620, Promega) was used according to manufacturer’s instructions. Briefly, 24 h after transfection in white 96-well culture plates, the medium was replaced with 70 μL of the fresh medium and assay was performed after an additional 24 h. The 96-well plates and the reagents were equilibrated to room temperature and 70 μL ONE-Glo™ EX was added to the culture medium. The samples were incubated for 10 min and Firefly luminescence was measured with a plate reader (GloMax® Discover System, Promega). For measuring NLuc luciferase activity, 70 μL of NanoDLR™ Stop& Glo® Reagent was added to each well, and the luminescence was measured after 20 min.
Klotho promoter-induced NLuc expression was measured in a NLuc knock-in HEK293 cell line using the Nano-Glo Luciferase Assay System (cat. N1110, Promega) according to the manufacturer’s instructions. Briefly, 24 h after transfection in white 96-well culture plates, the medium was replaced with 70 μL of the fresh medium and assay was performed after an additional 24 h. The 96-well plates and reagents were equilibrated to room temperature and 70 μL of Nano-Glo® Luciferase Assay Reagent was added to the culture medium. The samples were incubated for 10 min and the luminescence was measured using a plate reader (GloMax® Discover System, Promega).
qPCR Experiments and Analysis
Forty-eight hours after transfection, total RNA was isolated using the RNeasy mini plus Kit (QIAGEN) and 1 μg of total RNA was reversed transcribed using the SuperScript™ VILO™ cDNA Synthesis Kit according to the manufacturer’s instructions (cat. 11754050, ThermoFisher scientific). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was carried out for all genes of interest in each sample using human TaqMan Gene Expression Assays (Life Technologies): Klotho (Assay ID Hs00934627_m1 FAM); Peptidylprolyl isomerase A (PPIA) (Assay ID Hs04194521_s1; FAM) and beta actin (ActinB) (Assay ID Hs01060665_g1; VIC), on a BioRad 7900HT Real-Time PCR system using Fast Advanced Master Mix (Life Technologies), according to the manufacturer’s protocol. The Klotho transcript was normalized to PPIA and ActinB that were used as endogenous controls. Samples were run in triplicates at 1 μg of cDNA per reaction. The presence or absence of transcripts was assessed by whether a critical threshold (CT) value was determined or undetermined, respectively, at the threshold chosen by BioRad software v2.4. To normalize sample input, ΔCT values were calculated for each gene. Data were analyzed further by the ΔΔCt method, and fold changes in Klotho gene expression were determined by Gene Expression Module of CSX Manager software (BioRad), and a p value ≤ 0.05 was considered significant.
Protein Western Blotting was performed as described (Chen et al. 2007). Protein expression in Western blots was assessed and normalized by densitometry using ImageJ.
The antibodies used were for anti-Klotho (KO603, clone number KM2076, 1:500, Transgenic) and anti-Actin (1:1000, Cell Signaling, Danvers, MA).
For Western Blotting and Luciferase assay, the significance was calculated using the traditional Student’s t test. Quantitative data are expressed as the means ± SD. Statistical comparisons between experimental groups were made using the two-tailed, unpaired Student’s t test. Probability values of p < 0.05 were considered significant.
sgRNA target site selection
Evaluation of Klotho gene activation using a dual luciferase coincidence reporter system
Evaluation of Klotho Gene Activation Using a CRISPR NLuc Knock-in HEK293 cell line
Evaluation of Klotho Gene Activation in HK-2 and SY5Y Cells
In this study, we upregulate Klotho gene expression using a CRISPR-dCas9 SAM complex to investigate whether we could employ sgRNA targeting of the Klotho promoter region for efficient transcriptional activation of the Klotho gene. We identified two sgRNAs that can effectively enhance Klotho gene expression on the gene and protein level using three different assessments: a FLuc and NLuc coincidence reporter system, a NLuc luciferase knock-in in Klotho 3′-UTR using CRISPR genomic editing, and two cell lines that endogenously express Klotho: the human neuronal cell line SY5Y, in which the levels of Klotho are detectable by qPCR, but not detectable at the protein level, and a human kidney cell line HK-2 that endogenously expresses Klotho in an amount sufficient to be detected on the mRNA and protein levels. It is not surprising that we were unable to detect Klotho in SY5Y on the protein level, but on the mRNA level only: the Klotho transcript in SY5Y cells reaches a critical threshold (CT) value at around the 32nd cycle, providing evidence for the low abundance in these cells, which we have observed with other cell lines where Klotho is not detectable with the existent antibodies by Western blot. In contrast, in HK-2 cells, the Klotho transcript crosses a critical threshold (CT) value at the 26th cycle, and that explains the protein detection on Western blot.
Potential applications of CRISPR/RNA-guided genomic editing are diverse across many areas of science and biotechnology. CRISPR/dCas9 technology enables inexpensive and high-throughput interrogation of gene function, likely due to the simplicity, high efficiency, and versatility of the system. In this study, we used CRISPR technology to activate Klotho gene expression, and monitor its activation with a P2A-NLuc CRISPR knock-in cell line. The precise homology-directed repair pathway allows insertion of NLuc reporter in the Klotho 3′-UTR and monitor of Klotho gene transcript from the endogenous Klotho promoter using ribosome re-entry via the P2A sequence. The knock-in line has potential to be used for high-throughput drug screening to study Klotho gene regulation in a more physiologic system.
Taking advantage of the therapeutic potential of secreted proteins, such as Klotho, poses particular challenges for translational neurology, because of the profound difficulty in delivering these proteins across the blood-brain barrier (BBB). Another way to address this issue is by identifying small molecule compounds that are able to penetrate the BBB and stimulate the production of Klotho within the brain as a possible pathway in drug discovery. The luciferase reporter is commonly used in high-throughput screens to identify compounds that increase gene expression. However, many compound libraries contain molecules that may activate the reporter, either luciferase or other reporters used in screens (Hasson et al. 2015). These compounds present a potential challenge in the gain-of-signal assays since by binding reporters they increase the half-life of the reporter, producing a higher signal, which is translated to an activation-like signal in the cell based assays (Cheng and Inglese 2012; Hasson et al. 2015).
In our previously published work, we conducted a high-throughput screen (HTS) to identify compounds that activate Klotho transcription using the Klotho 1.8 kb promoter to drive expression of firefly luciferase; however, the rate of false positive hits increasing the luciferase signal independently of Klotho promoter was high and the discrimination between the real and false positive hits was challenging (King et al. 2012). This limitation can be mitigated with a “coincidence reporter”: a system that allows expression of both firefly luciferase and NanoLuc® Luciferase from the same mRNA transcript. The stoichiometric expression of both luciferases is achieved by use of the P2A sequence, which promotes a ribosomal skip and expression of the two unfused enzymes with distinct compound interaction profiles. We are currently utilizing this system for a new HTS of a library of 50,000 compounds. Transfections using Egr-1 transcription factor, known to activate Klotho transcription, provide a reliable positive control for the assay leading to consistent and significant increase in the expression of both luciferases. In this current assay we are able to identify false hits caused by direct interaction with one or the other luciferases and to distinguish them from true hits that show a similar response for both luciferases, adding reproducibility to the assay.
Compared to the CRISPR NLuc knock-in HEK293 cell line, the 4 kb Klotho promoter coincidence reporter system appears more suitable for HTS purpose. The signal observed in the 4 kb Klotho promoter coincidence reporter system is higher than in the NLuc knock-in line likely because the endogenous Klotho promoter is not very active likely due to the presence of repressors or methylation since the Klotho gene is mostly expressed in specific tissues such as kidney and brain and is downregulated with age and in disease.
Modulation of the levels of one protein, Klotho, in mice results in dramatic changes in lifespan and cognition (Dubal et al. 2015; Kuro-o et al. 1997; Massó et al. 2017). Knocking-out the gene, while not affecting development or adolescence, results in premature death in adulthood (Kuro-o et al. 1997). In contrast, overexpression of Klotho increases lifespan by ~ 30% over normal mice (Kurosu et al. 2005). In the CNS Klotho has pleiotropic functions, it is neuroprotective and anti-oxidative (Zeldich et al. 2014) and is involved in oligodendrocytes maturation and myelination in vivo and in vitro (Chen et al. 2015; Chen et al. 2013; Zeldich et al. 2015). Klotho overexpression reduces cognitive deficits in a mouse model of Alzheimer’s disease and enhances cognition in humans and mice (Dubal et al. 2014; Dubal et al. 2015; Massó et al. 2017). Increasing Klotho levels in the brain would have a beneficial effect to prevent cognitive impairment in the aged population and protect against neurodegeneration. In a high-throughput screen of small molecules that would enhance Klotho expression, we have identified a number of promising compounds that elevate Klotho expression at the RNA, protein, and functional levels (Abraham et al. 2012; King et al. 2012). However, most compounds have inevitable off-target effects and cytotoxicity. Here we provide information of activation of the anti-aging gene Klotho via CRISPR/RNA-guided transcription activation. This system presents an alternative assay for identifying specific Klotho gene activation. The results described here are valuable for the study of Klotho gene regulation and have great potential in gene therapeutics. The technology could be tested in vivo in animal models of various diseases, including the currently untreatable multiple sclerosis and neurodegenerative diseases such as Alzheimer’s disease.
We thank Dr. Jason Nasse for reading and helpful suggestions for the manuscript.
This work was supported by NIH grants R56 AG051638, R44 AG053084 and R01 AG048927.
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