Hypercompact adenine base editors based on transposase B guided by engineered RNA.

Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here we suggest a new technology whereby the engineered guide RNAs also manifest high-efficiency programmable endonuclease activity for TnpB. We have termed this technology TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors (ABEs). A Tad-Tad mutant (V106W, D108Q) dimer fused to the C terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded with engineered variants of TnpB or optimized deaminases. Delivery of TaRGET-ABE also ensured potent A-to-G conversion rates in mammalian genomes. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by adeno-associated viruses, thereby harnessing the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene therapy.

Unbiased investigation of specificities of prime editing systems in human cells.

Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.

Depletion of ST6GALNACIII retards A549 non-small cell lung cancer cell proliferation by downregulating transferrin receptor protein 1 expression.

Alterations in sialylation of terminal residues of glycoproteins have been implicated in forming tumor-associated glycans. ST6GALNAC transfers sialyl moiety to N-acetylgalactosamine residue via α2,6 linkage. Although the oncogenic characteristics of ST6GALNACI or II have been demonstrated in various cancer cells, the impact of ST6GALNACIII on tumor progression remains undefined. In this study, we evaluated the effect of ST6GALNACIII knockdown on the growth of A549 non-small cell lung cancer cells. ST6GALNACIII depletion resulted in significant retardation in growth of A549 cells under various culture conditions, including collagen-supported 3D culture and anchorage-independent soft agar culture conditions. Liquid chromatography with tandem mass spectrometry revealed that two glycopeptides of transferrin receptor protein 1 (TFR1) containing N-acetylhexosamine-sialic acid were not detected in ST6GALNACIII-depleted A549 cells compared with control cells. Subsequent lectin binding assay, western blotting, and real-time RT-PCR indicated that TFR1 sialylation was not significantly changed, but TFR1 protein and mRNA expressions were decreased after ST6GALNACIII knockdown. However, cell growth retardation by ST6GALNACIII knockdown was partially rescued by TFR1 overexpression. Additionally, TFR1 mRNA degradation was accelerated following ST6GALNACIII knockdown with concomitant reduction in mRNA levels of iron regulatory protein 1 and 2, the upstream regulators of TFR1 mRNA stability. Therefore, our results indicated an important role of ST6GALNACIII in promoting A549 cell growth through quantitative regulation of TFR1 expression and provided therapeutic implications for ST6GALNACIII targeting in tumor growth suppression in vivo.

One-step genotyping method in CRISPR based on short inner primer-assisted, tetra primer-paired amplifications.

Base editors and prime editors induce precise DNA modifications over one or several nucleotides in eukaryotic cells. The T7E1 assay has been widely adopted for the assessment of genome editing, but it has several limitations in the applications for prime editing and base editing due to low sensitivity, inaccuracy and additional disadvantages. Here, we propose a short inner primer-assisted, tetra primer-paired amplification (SIPATA) method as an alternative to T7E1 analysis. SIPATA is a PCR-based method in which two long outer and two short (15 nt) inner primers are used for the amplification of a specific genotype in the presence of Hot start-Taq. One of the inner primers carries a 3'-terminally wild-type nucleotide sequence, and the other carries a post-editing sequence. Under optimized conditions, SIPATA enabled sensitive and accurate genotyping of single-nucleotide conversions by base editors and prime editors. Furthermore, SIPATA could be applied to trace low levels of DNA modifications achieved by HDR-mediated gene correction or chimerism during the generation of model animals. Multiplexed genotyping was also possible without compromising those multifaceted analytical advantages of SIPATA. Our findings demonstrate that SIPATA offers a robust, fast and sensitive genotyping platform for single-nucleotide variations in a variety of CRISPR applications.

Selective Identification of α-Galactosyl Epitopes in N-Glycoproteins Using Characteristic Fragment Ions from Higher-Energy Collisional Dissociation.

The α-galactosyl epitope is a terminal N-glycan moiety of glycoproteins found in mammals except in humans, and thus, it is recognized as an antigen that provokes an immunogenic response in humans. Accordingly, it is necessary to analyze the α-galactosyl structure in biopharmaceuticals or organ transplants. Due to an identical glycan composition and molecular mass between α-galactosyl N-glycans and hybrid/high-mannose-type N-glycans, it is challenging to characterize α-galactosyl epitopes in N-glycoproteins using mass spectrometry. Here, we describe a method to identify α-galactosyl N-glycopeptides in mice glycoproteins using liquid chromatography with tandem mass spectrometry with higher-energy collisional dissociation (HCD). The first measure was an absence of [YHM] ion peaks in the HCD spectra, which was exclusively observed in hybrid and/or high-mannose-type N-glycopeptides. The second complementary criterion was the ratio of an m/z 528.19 (Hex2HexNAc1) ion to m/z 366.14 (Hex1HexNAc1) ion (Im/z528/Im/z366). The measure of [Im/z528/Im/z366 > 0.3] enabled a clear-cut determination of α-galactosyl N-glycopeptides with high accuracy. In Ggta1 knockout mice, we could not find any α-galactosyl N-glycoproteins identified in WT mice plasma. Using this method, we could screen for α-galactosyl N-glycoproteins from mice spleen, lungs, and plasma samples in a highly sensitive and specific manner. Conclusively, we suggest that this method will provide a robust analytical tool for determination of α-galactosyl epitopes in pharmaceuticals and complex biological samples.

Effect of BIS depletion on HSF1-dependent transcriptional activation in A549 non-small cell lung cancer cells.

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

Semi-quantitative measurement of a specific glycoform using a DNA-tagged antibody and lectin affinity chromatography for glyco-biomarker development.

Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates.

Application of cancer-associated glycoforms and glycan-binding probes to an in vitro diagnostic multivariate index assay for precise diagnoses of cancer.

Personalized medicine has emerged as a widely accepted trend in medicine for the efficacious and safe treatment of various diseases. It covers every medical treatment tailored according to various properties of individuals. Cancer-associated glycosylation mirrors cancer states more precisely, and this "sweet side of cancer" is thus intended to spur the development of an advanced in vitro diagnostic system. The changes of glyco-codes are often subtle and thus not easy to trace, thereby making it difficult to discriminate changes from various compounding factors. Special glycan-binding probes, often lectins, can be paired with aglycosylated antibodies to enable quantitative and qualitative measurements of glycoforms. With the in vitro diagnosis multivariate index assay (IVDMIA) considered to be capable of yielding patient-specific results, the combinatorial use of multiple glycoproteins may be a good modality to ensure disease-specific, personalized diagnoses.

Identification of Mycobacterial Antigens in Human Urine by Use of Immunoglobulin G Isolated from Sera of Patients with Active Pulmonary Tuberculosis.

Point-of-care (POC) diagnostic testing of tuberculosis (TB) is a tremendous unmet need. In this study, four urinary mycobacterial antigens were identified through two independent approaches using IgG capture and immunodepletion methods. Among these, ModC was validated by a multiple reaction monitoring (MRM) method. As expected, the biomarkers elevated the clinical validity of TB diagnosis when combined with preexisting markers.

Hepatitis B virus X protein specially regulates the sialyl lewis a synthesis among glycosylation events for metastasis.

BACKGROUND: The metastasis of hematogenous cancer cells is associated with abnormal glycosylation such as sialyl lewis antigens. Although the hepatitis B virus X protein (HBx) plays important role in liver disease, the precise function of HBx on aberrant glycosylation for metastasis remains unclear. METHODS: The human hepatocellular carcinoma tissues, HBx transgenic mice and HBx-transfected cells were used to check the correlation of expressions between HBx and Sialyl lewis antigen for cancer metastasis. To investigate whether expression levels of glycosyltransferases induced in HBx-transfected cells are specifically associated with sialyl lewis A (SLA) synthesis, which enhances metastasis by interaction of liver cancer cells with endothelial cells, ShRNA and siRNAs targeting specific glycosyltransferases were used. RESULTS: HBx expression in liver cancer region of HCC is associated with the specific synthesis of SLA. Furthermore, the SLA was specifically induced both in liver tissues from HBx-transgenic mice and in in vitro HBx-transfected cells. HBx increased transcription levels and activities of α2-3 sialyltransferases (ST3Gal III), α1-3/4 fucosyltransferases III and VII (FUT III and VII) genes, which were specific for SLA synthesis, allowing dramatic cell-cell adhesion for metastatic potential. Interestingly, HBx specifically induced expression of N-acetylglucosamine-ß1-3 galactosyltransferase V (ß1-3GalT 5) gene associated with the initial synthesis of sialyl lewis A, but not ß1-4GalT I. The ß1-3GalT 5 shRNA suppressed SLA expression by HBx, blocking the adhesion of HBx-transfected cells to the endothelial cells. Moreover, ß1-3GalT 5 silencing suppressed lung metastasis of HBx-transfected cells in in vivo lung metastasis system. CONCLUSION: HBx targets the specific glycosyltransferases for the SLA synthesis and this process regulates hematogenous cancer cell adhesion to endothelial cells for cancer metastasis.

PHLPP1 regulates contact inhibition by dephosphorylating Mst1 at the inhibitory site.

Contact inhibition has been largely elusive despite that a loss of contact inhibition is a critical event for cancer development and progression. Here, we report that PHLPP1 is a binding protein for Mst1 and it modulates the Hippo pathway by dephosphorylating Mst1 at the inhibitory Thr(387) of Mst1. Yap1 was localized predominantly in the nucleus but marginally in the cytoplasm in HeLa cells under sparse conditions, whereas the functional protein was more directed to sequestration in the cytoplasm under dense environments. Furthermore, loss of PHLPP1 resulted in a failure of the apoptotic control. It is interesting that down-regulated expression of PHLPP1 appears to mimic the loss of contact inhibition, a hallmark of cancer.

Overexpression and ß-1,6-N-acetylglucosaminylation-initiated aberrant glycosylation of TIMP-1: a "double whammy" strategy in colon cancer progression.

There has been ongoing debate over whether tissue inhibitor of metalloproteinase-1 (TIMP-1) is pro- or anti-oncogenic. We confirmed that TIMP-1 reinforced cell proliferation in an αvß3 integrin-dependent manner and conferred resistance against cytotoxicity triggered by TNF-α and IL-2 in WiDr colon cancer cells. The cell-proliferative effects of TIMP-1 contributed to clonogenicity and tumor growth during the onset and early phase of tumor formation in vivo and in vitro. However, mass-produced TIMP-1 impeded further tumor growth by tightly inhibiting the activities of collagenases, which are critical for tumor growth and malignant transformation. Tumor cells could overcome this impasse by overexpression of N-acetylglucosaminyltransferase V, which deteriorates TIMP-1 into an aberrant glycoform. The aberrant glycoform of TIMP-1 was responsible for the mitigated inhibition of collagenases. The outbalanced activities of collagenases can degrade the basement membrane and the interstitial matrix, which act as a physical barrier for tumor growth and progression more efficiently. The concomitant overexpression of TIMP-1 and N-acetylglucosaminyltransferase V enabled WiDr cells to show a higher tumor growth rate as well as more malignant behaviors in a three-dimensional culture system.

Inhibition of MKK7-JNK by the TOR signaling pathway regulator-like protein contributes to resistance of HCC cells to TRAIL-induced apoptosis.

BACKGROUND & AIMS: The TOR signaling pathway regulator-like (TIPRL) protein, the mammalian ortholog of yeast TIP41, was identified in an expression profiling screen for factors that regulate human liver carcinogenesis. We investigated the role of human TIPRL protein in hepatocellular carcinoma (HCC). METHODS: We measured the level of TIPRL in HCC and adjacent nontumor tissues from patients. We used small interfering RNAs and zebrafish to study the function of TIPRL. We used annexin V propidium iodide staining and immunoblot analyses to measure apoptosis and activation of apoptotic signaling pathways. We used confocal microscopy, coimmunoprecipitation, and glutathione-S transferase pull-down analyses to determine interactions among mitogen-activated protein kinase kinase 7 (MKK7 or MAP2K7), TIPRL, and the protein phosphatase type 2A (PP2Ac). We studied the effects of TIPRL in tumor xenografts in mice. RESULTS: Levels of TIPRL were higher in HCC tissues and cell lines than nontumor tissues and primary hepatocytes. Knockdown of tiprl expression in zebrafish led to large amounts of apoptosis throughout the embryos. Incubation of HCC cells, but not primary human hepatocytes, with small interfering RNA against TIPRL (siTIPRL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) caused prolonged activation (phosphorylation) of MKK7 and c-Jun N-terminal kinase (JNK) and led to apoptosis, indicated by cleavage of procaspase-8,-3 and of poly-(adenosine diphosphate-ribose) polymerase. TIPRL bound to MKK7 and PP2Ac and promoted the interaction between MKK7 and PP2Ac. In mice, injection of HCC xenograft tumors with siTIPRL and TRAIL led to tumor apoptosis and regression. CONCLUSIONS: TIPRL is highly up-regulated in human HCC samples and cell lines, compared with noncancerous liver tissues. TIPRL prevents prolonged activation of MKK7 and JNK and TRAIL-induced apoptosis by mediating the interaction between MKK7 and PP2Ac.

N-Acetylglucosaminyltransferase V triggers overexpression of MT1-MMP and reinforces the invasive/metastatic potential of cancer cells.

N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes the formation of a ß1,6-N-acetylglucosamine (GlcNAc) side chain to a core mannosyl residue in N-linked glycoproteins. Besides its direct function of producing aberrant glycoproteins, it promotes cancer progression by its involvement in the stimulation of oncoproteins. Herein, we report that GnT-V guided the transcriptional activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) in cancer cells. The activated MT1-MMP expression had dual effects on cancer progression. It not only promoted proteolytic activity for cancer cells per se, but also led to the activation of MMP-2. Consequently, the activation of the two MMPs triggered by GnT-V intensified the invasive potential. A quantitative analysis using clinical tissues revealed a relatively strong correlation between GnT-V overexpression and MT1-MMP upregulation. In this study, we report for the first time that GnT-V directs cancer progression by modulating MMPs in cancer.

A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry.

A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states.

SF3B4 Depletion Retards the Growth of A549 Non-Small Cell Lung Cancer Cells via UBE4B-Mediated Regulation of p53/p21 and p27 Expression.

Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The in vivo expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.

Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.

Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

Defect in cytosolic Neu2 sialidase abrogates lipid metabolism and impairs muscle function in vivo.

Sialic acid (SA) is present in glycoconjugates and important in cell-cell recognition, cell adhesion, and cell growth and as a receptor. Among the four mammalian sialidases, cytosolic NEU2 has a pivotal role in muscle and neuronal differentiation in vitro. However, its biological functions in vivo remain unclear due to its very low expression in humans. However, the presence of cytoplasmic glycoproteins, gangliosides, and lectins involved in cellular metabolism and glycan recognition has suggested the functional importance of cytosolic Neu2 sialidases. We generated a Neu2 knockout mouse model via CRISPR/Cas9-mediated genome engineering and analyzed the offspring littermates at different ages to investigate the in vivo function of cytosolic Neu2 sialidase. Surprisingly, knocking out the Neu2 gene in vivo abrogated overall lipid metabolism, impairing motor function and leading to diabetes. Consistent with these results, Neu2 knockout led to alterations in sialylated glycoproteins involved in lipid metabolism and muscle function, as shown by glycoproteomics analysis.

Galectin-3 binding protein promotes cell motility in colon cancer by stimulating the shedding of protein tyrosine phosphatase kappa by proprotein convertase 5.

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.