Qiurong Ding

Enhanced Efficiency of Human Pluripotent Stem Cell Genome Editing through Replacing TALENs with CRISPRs

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are new classes of genome-editing tools that target desired genomic sites in mammalian cells (Miller et al., 2011; Hockemeyer et al., 2011; Cong et al., 2013; Mali et al., 2013; Jinek et al., 2013). TALENs bind as a pair around a genomic site, in which a double-strand break (DSB) is introduced by a dimer of FokI nuclease domains. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence (“protospacer”) beginning with G and immediately preceding an NGG motif recognized by Cas9—constituting a G(N)19NGG target DNA sequence—resulting in a DSB three nucleotides upstream of the NGG motif (Jinek et al., 2012). However it is generated, the DSB instigates either non-homologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations (indels) that knock out gene alleles, or homology-directed repair (HDR), which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. We recently reported the use of a TALEN genome-editing system to rapidly and efficiently generate mutant alleles of 15 different genes in human pluripotent stem cells (hPSCs) as a means of performing rigorous disease modeling (Ding et al., 2013); the proportions of clones bearing at least one mutant alelle ranged from 2%–34%. Although one example of the use of CRISPRs in hPSCs has been reported (Mali et al., 2013), the efficiency of allele targeting was only 2%–4% (albeit in unsorted cells, in contrast to our system; see below). We sought to compare the relative efficacies of CRISPRs and TALENs targeting the same genomic sites in the same hPSC lines with the use of the same delivery platform as we described previously (Ding et al., 2013). In the TALEN genome-editing system, we used the CAG promoter to cotranslate (via a viral 2A peptide) each TALEN with green fluorescent protein (GFP) or red fluorescent protein (RFP). For CRISPRs, we subcloned a human codon-optimized Cas9 gene with a C-terminal nuclear localization signal (Mali et al., 2013) into the same CAG expression plasmid with GFP, and we separately expressed the guide RNA (gRNA) from a plasmid with the human U6 polymerase III promoter (Mali et al., 2013). The 20-nucleotide protospacer sequence for each gRNA was introduced using polymerase chain reaction (PCR)-based methods. Whether using TALENs or CRISPRs, equal amounts of the two plasmids were co-electroporated into hPSCs—either 25 μg of each plasmid, or 15 μg of each plasmid along with 30 μg of a DNA repair template if attempting knock-in—followed by fluorescence-activated cell sorting (FACS) after 24–48 hours, clonal expansion of single cells, and screening for mutations at the genomic target site via PCR. We designed gRNAs matching G(N)19NGG sequences in seven loci in six genes—AKT2, CELSR2, CIITA, GLUT4, LINC00116, and SORT1—that we had previously successfully targeted with TALENs (Ding et al., 2013) and one locus, in LDLR, that we had not. We found that in our system CRISPRs consistently and substantially outperformed TALENs across loci and hPSC lines (see Table S1). The TALENs yielded clones with at least one mutant allele at efficiencies of 0%–34%, but matched CRISPRs yielded mutant clones at efficiencies of 51%–79% (Table S1). Just as with TALENs, CRISPRs produced a variety of indels of sizes ranging from one nucleotide to several dozen nucleotides in size, centered on the predicted cleavage sites, suggesting that NHEJ mutagenesis occurs in the same way regardless of whether CRISPRs or TALENs are used. We also found that CRISPRs readily generated homozygous mutant clones (7%–25% of all clones; Table S1) as discerned by sequencing. We also attempted to knock in E17K mutations into AKT2 using a 67-nucleotide single-stranded DNA oligonucleotide as previously described (Ding et al., 2013). Although the predicted CRISPR cleavage site lay 11 and 13 nucleotides from the point mutations, respectively, the CRISPR yielded knock-in clones at a rate of 11%, whereas TALENs yielded only 1.6% (Table S1). We speculate that the superior performance of CRISPRs in our system is due to the Cas9 protein being more highly expressed and better tolerated than TALENs in hPSCs, as we routinely observed earlier (<24 hours vs. 48 hours) and more robust (5%–10% of cells vs. <1%–2% of cells) GFP expression following electroporation. Other factors may include intrinsic DNA-unwinding activity of Cas9 and impaired TALEN binding on methylated DNA. It is possible that further optimization of the TALEN system that we developed could improve its efficiency and reduce the differential that we observe. Two potential disadvantages of CRISPRs are worth noting. First, the requirement for a G(N)19NGG target sequence somewhat limits site selection. Because either DNA strand can be targeted, a target sequence occurs on average every 32 basepairs. This is no barrier for gene knockout, where any coding sequence can be targeted, but it may present difficulties when trying to knock in or correct a mutation at a specific location. However, the requirement for a G at the start of the protospacer is dictated by the use of the U6 promoter to express the gRNA, and alternative CRISPR/Cas systems can relieve this requirement (Cong et al., 2013). Second, the extent of CRISPR off-target effects remains to be defined. Previous analyses have suggested that one-nucleotide mismatches in the first half of the protospacer are better tolerated than mismatches in second half (Jinek et al., 2012; Cong et al., 2013). None of the genomic sequences we targeted with CRISPRs have perfectly-matched or one-mismatch sequences elsewhere in the genome. For the AKT2 sequence, there is a two-mismatch sequence differing at nucleotides 1 and 3, in the more “tolerant” half of the protospacer; we obtained zero clones with mutations at this potential off-target site, as compared to 61% at the on-target site (Table S1), suggesting that at least in this instance off-target effects are not likely to be a significant concern. Judicious selection of target sites may well be able to minimize systematic off-target effects. Nevertheless, clear-cut determination of the relative risk for both TALEN- and CRISPR-based approaches will require a systematic analysis. It is important to highlight that all of these genome-editing technology approaches are still very much in development, and more detailed and comprehensive studies will be needed to determine their relative merits in different experimental circumstances. From a practical standpoint, CRISPRs are easier to implement than TALENs, as each TALEN pair must be constructed de novo, whereas for CRISPRs the Cas9 component is fixed and the gRNA requires only swapping of the 20-nucleotide protospacer. Given this consideration and our observations of substantially increased efficiency through replacing TALENs with CRISPRs in an otherwise identical system, we would suggest that CRISPRs might well prove to be a very powerful and broadly applicable tool for the stem cell community.

Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing

Rationale: Individuals with naturally occurring loss-of-function proprotein convertase subtilisin/kexin type 9 (PCSK9) mutations experience reduced low-density lipoprotein cholesterol levels and protection against cardiovascular disease. Objective: The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo. Methods and Results: We used adenovirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that <3 to 4 days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein receptor levels, and decreased plasma cholesterol levels (by 35–40%). No off-target mutagenesis was detected in 10 selected sites. Conclusions: Genome editing with the CRISPR–CRISPR-associated 9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.

Hepatic sortilin regulates both apolipoprotein B secretion and LDL catabolism

Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo—Brief Report

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.

Interleukin-6 stimulates aerobic glycolysis by regulating PFKFB3 at early stage of colorectal cancer

Chronic inflammation is a well-known etiological factor for colorectal cancer (CRC) and cancer cells are known to preferentially metabolize glucose through aerobic glycolysis. However, the connection between chronic inflammation and aerobic glycolysis in the development of CRC is largely unexplored. The present study investigated whether interleukin-6 (IL-6), a pro-inflammatory cytokine, promotes the development of CRC by regulating the aerobic glycolysis and the underlying molecular mechanisms. In colitis-associated CRC mouse, anti-IL-6 receptor antibody treatment reduced the incidence of CRC and decreased the expression of key genes in aerobic glycolysis, whereas the plasma concentrations of glucose and lactate were not affected. Consistently, IL-6 treatment stimulated aerobic glycolysis, upregulated key genes in aerobic glycolysis and promoted cell proliferation and migration in SW480 and SW1116 CRC cells. 6-phoshofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) was the most downregulated gene by anti-IL-6 receptor antibody in colorectal adenoma tissues. Further analysis in human samples revealed overexpression of PFKFB3 in colorectal adenoma and adenocarcinoma tissues, which was also associated with lymph node metastasis, intravascular cancer embolus and TNM stage. In addition, the effect of IL-6 on CRC cells can be abolished by knocking down PRKFB3 through siRNA transfection. Our data suggest that chronic inflammation promotes the development of CRC by stimulating aerobic glycolysis and IL-6 is functioning, at least partly, through regulating PFKFB3 at early stage of CRC.

Genome engineering of stem cell organoids for disease modeling

Precision medicine emerges as a new approach that takes into account individual variability. Successful realization of precision medicine requires disease models that are able to incorporate personalized disease information and recapitulate disease development processes at the molecular, cellular and organ levels. With recent development in stem cell field, a variety of tissue organoids can be derived from patient specific pluripotent stem cells and adult stem cells. In combination with the state-of-the-art genome editing tools, organoids can be further engineered to mimic disease-relevant genetic and epigenetic status of a patient. This has therefore enabled a rapid expansion of sophisticated in vitro disease models, offering a unique system for fundamental and biomedical research as well as the development of personalized medicine. Here we summarize some of the latest advances and future perspectives in engineering stem cell organoids for human disease modeling.

Titanium dioxide nanoparticles prime a specific activation state of macrophages

Abstract Titanium dioxide nanoparticles (TiO2 NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO2 NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO2 NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO2 NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO2 NPs (10 and 50 nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO2 NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO2 NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO2 NPs. These results demonstrate that TiO2 NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.

RIP1 kinase activity-dependent roles in embryonic development of Fadd-deficient mice

RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd−/− embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1K45A/K45A mouse line as previously reported and a novel Rip1Δ/Δ mouse line with an altered P-loop in the kinase domain. While RIP1K45A could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1Δ rescued lethality of Fadd−/− mice at E11.5 and Fadd−/−Rip1Δ/Δ mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1K45A/K45A cells, Rip1Δ/Δcells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.

Interleukin-6 promotes tumor progression in colitis‑associated colorectal cancer through HIF-1α regulation

Interleukin-6 (IL-6) is a well-known etiological factor of colitis-associated colorectal cancer (CAC) and has a significant role in CAC progression. In addition, hypoxia-inducible factor 1α (HIF-1α) serves a primary role in the progression of CAC. However, the association between IL-6 and HIF-1α during the progression of CAC remains unclear. To investigate this association, the present study induced CAC in a mouse model using azoxymethane and dextran sulfate sodium. In addition, an anti-IL-6 receptor antibody was used to inhibit IL-6. In this model, anti-IL-6 receptor antibody treatment significantly inhibited the development of CAC and the expression of HIF-1α, in colorectal adenomas and adenocarcinomas. In patients with CAC, the HIF-1α gene was demonstrated to be overexpressed in tumor tissue compared with adjacent non-malignant tissue. Furthermore, HIF-1α mRNA expression was positively correlated with serum IL-6 concentration. The results of the present study suggest that IL-6 promotes CAC progression, in the early stage of the disease, through HIF-1α regulation.