Perturbation of Gene Regulation by Genome Editing.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor. Here we describe a set of protocols for using the catalytically inactive dead Cas9 (dCas9)-based tools, including the bipartite super repressor consisting of the KRAB and MeCP2 domains, to achieve efficient and scalable gene silencing in mammalian cells.

Variable phenotypes and penetrance between and within different zebrafish ciliary transition zone mutants.

Meckel syndrome, nephronophthisis, Joubert syndrome and Bardet-Biedl syndrome are caused by mutations in proteins that localize to the ciliary transition zone (TZ). The phenotypically distinct syndromes suggest that these TZ proteins have differing functions. However, mutations in a single TZ gene can result in multiple syndromes, suggesting that the phenotype is influenced by modifier genes. We performed a comprehensive analysis of ten zebrafish TZ mutants, including mks1, tmem216, tmem67, rpgrip1l, cc2d2a, b9d2, cep290, tctn1, nphp1 and nphp4, as well as mutants in ift88 and ift172. Our data indicate that variations in phenotypes exist between different TZ mutants, supporting different tissue-specific functions of these TZ genes. Further, we observed phenotypic variations within progeny of a single TZ mutant, reminiscent of multiple disease syndromes being associated with mutations in one gene. In some mutants, the dynamics of the phenotype became complex with transitory phenotypes that are corrected over time. We also demonstrated that multiple-guide-derived CRISPR/Cas9 F0 'crispant' embryos recapitulate zygotic null phenotypes, and rapidly identified ciliary phenotypes in 11 cilia-associated gene candidates (ankfn1, ccdc65, cfap57, fhad1, nme7, pacrg, saxo2, c1orf194, ttc26, zmynd12 and cfap52).

Loss of IRF2BPL impairs neuronal maintenance through excess Wnt signaling.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.

Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis.

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

Synthetic immunomodulation with a CRISPR super-repressor in vivo.

Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors-heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)-are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.

Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.

Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD.

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity.

It has been possible to create tools to predict single guide RNA (sgRNA) activity in the CRISPR/Cas9 system derived from Streptococcus pyogenes due to the large amount of data that has been generated in sgRNA library screens. However, with the discovery of additional CRISPR systems from different bacteria, which show potent activity in eukaryotic cells, the approach of generating large data sets for each of these systems to predict their activity is not tractable. Here, we present a new guide RNA tool that can predict sgRNA activity across multiple CRISPR systems. In addition to predicting activity for Cas9 from S. pyogenes and Streptococcus thermophilus CRISPR1, we experimentally demonstrate that our algorithm can predict activity for Cas9 from Staphylococcus aureus and S. thermophilus CRISPR3. We also have made available a new version of our software, sgRNA Scorer 2.0, which will allow users to identify sgRNA sites for any PAM sequence of interest.

Genome-wide Association Studies Identify Genetic Loci Associated With Albuminuria in Diabetes.

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and are associated with an increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (P = 2.4 × 10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. Single nucleotide polymorphisms at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in the average UACR per minor allele was 21% for HS6ST1 (P = 6.3 × 10(-7)) and 13% for RAB38/CTSC (P = 5.8 × 10(-7)). Experiments using streptozotocin-induced diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout versus control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared with control subjects. The loci identified here confirm known pathways and highlight novel pathways influencing albuminuria.

Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity.

Genome-wide association studies (GWAS) identify regions of the genome correlated with disease risk but are restricted in their ability to identify the underlying causative mechanism(s). Thus, GWAS are useful "roadmaps" that require functional analysis to establish the genetic and mechanistic structure of a particular locus. Unfortunately, direct functional testing in humans is limited, demonstrating the need for complementary approaches. Here we used an integrated approach combining zebrafish, rat, and human data to interrogate the function of an established GWAS locus (SHROOM3) lacking prior functional support for chronic kidney disease (CKD). Congenic mapping and sequence analysis in rats suggested Shroom3 was a strong positional candidate gene. Transferring a 6.1-Mb region containing the wild-type Shroom3 gene significantly improved the kidney glomerular function in FHH (fawn-hooded hypertensive) rat. The wild-type Shroom3 allele, but not the FHH Shroom3 allele, rescued glomerular defects induced by knockdown of endogenous shroom3 in zebrafish, suggesting that the FHH Shroom3 allele is defective and likely contributes to renal injury in the FHH rat. We also show for the first time that variants disrupting the actin-binding domain of SHROOM3 may cause podocyte effacement and impairment of the glomerular filtration barrier.

Refined mapping of a hypertension susceptibility locus on rat chromosome 12.

Previously, we found that transferring 6.1 Mb of salt-sensitive (SS) chromosome 12 (13.4-19.5 Mb) onto the consomic SS-12(BN) background significantly elevated mean arterial pressure in response to an 8% NaCl diet (178±7 versus 144±2 mm Hg; P<0.001). Using congenic mapping, we have now narrowed the blood pressure locus by 86% from a 6.1-Mb region containing 133 genes to an 830-kb region (chr12:14.36-15.19 Mb) with 14 genes. Compared with the SS-12(BN) consomic, the 830-kb blood pressure locus was associated with a ∆+15 mm Hg (P<0.01) increase in blood pressure, which coincided with elevated albuminuria (∆+32 mg/d; P<0.001), proteinuria (∆+48 mg/d; P<0.01), protein casting (∆+154%; P<0.05), and renal fibrosis (∆+79%; P<0.05). Of the 14 genes residing in the 830-kb locus, 8 were differentially expressed, and among these, Chst12 (carbohydrate chondroitin 4 sulfotransferase 12) was most consistently downregulated by 2.6- to 4.5-fold (P<0.05) in both the renal medulla and cortex under normotensive and hypertensive conditions. Moreover, whole genome sequence analysis of overlapping blood pressure loci revealed an ≈86-kb region (chr12:14 541 567-14 627 442 bp) containing single-nucleotide variants near Chst12 that are unique to the hypertensive SS strain when compared with the normotensive Brown Norway, Dahl salt-resistant, and Wistar-Kyoto strains. Finally, the 830-kb interval is syntenic to a region on human chromosome 7 that has been genetically linked to blood pressure, suggesting that insight gained from our SS-12(BN) congenic strain may be translated to a better understanding of human hypertension.

Renal medullary microRNAs in Dahl salt-sensitive rats: miR-29b regulates several collagens and related genes.

MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13(BN) rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13(BN) rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13(BN) rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13(BN) rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin beta1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13(BN) rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13(BN) rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3'-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13(BN) rats.