Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases
has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)
. To address this limitation, we engineered an enhanced ...Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants
to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation ...domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
Ovarian cancer is a lethal gynecological malignancy, and to improve survival, it is important to identify novel prognostic and therapeutic targets. In this study, we present a role for p21-activated ...kinase 4 (Pak4) in ovarian cancer progression. We show a significant association between increased expression of Pak4 and its activated form, phosphorylated (p)-Pak4 Ser⁴⁷⁴, with metastasis of ovarian cancers, shorter overall and disease-free survival, advanced stage and high-grade cancers, serous/clear cell histological subtypes, and reduced chemosensitivity. Pak4 overexpression was also observed in ovarian cancer cell lines. Pak4 and p-Pak4 expression were detected both in the nucleus and cytoplasm of ovarian cancer cells, in vitro as well as in vivo. Stable knockdown of Pak4 in ovarian cancer cell lines led to reduced cell migration, invasion, and proliferation, along with reduced c-Src, ERK1/2, and epidermal growth factor receptor (EGFR) activation and decreased matrix metalloproteinase 2 (MMP2) expression. Conversely, Pak4 overexpression promoted ovarian cancer cell migration and invasion in a c-Src, MEK-1, MMP2, and kinase-dependent manner, and induced cell proliferation through the Pak4/c-Src/EGFR pathway that controls cyclin D1 and CDC25A expression. Stable knockdown of Pak4 also impeded tumor growth and dissemination in nude mice. This report reveals the association between Pak4 and important clinicopathologic parameters, suggesting Pak4 to be a significant prognostic marker and potential therapeutic molecular target in ovarian cancer. The implied possible cross-talk between Pak4 and EGFR suggests the potential of dual targeting of EGFR and Pak4 as a unique therapeutic approach for cancer therapy.
Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting ...aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.
Hyperkalaemia is an electrolyte imbalance that impairs muscle function and myocardial excitability, and can potentially lead to fatal arrhythmias and sudden cardiac death. The prevalence of ...hyperkalaemia is estimated to be 6%–7% worldwide and 7%–10% in Asia. Hyperkalaemia frequently affects patients with chronic kidney disease, heart failure, and diabetes mellitus, particularly those receiving treatment with renin‐angiotensin‐aldosterone system (RAAS) inhibitors. Both hyperkalaemia and interruption of RAAS inhibitor therapy are associated with increased risks for cardiovascular events, hospitalisations, and death, highlighting a clinical dilemma in high‐risk patients. Conventional potassium‐binding resins are widely used for the treatment of hyperkalaemia; however, caveats such as the unpalatable taste and the risk of gastrointestinal side effects limit their chronic use. Recent evidence suggests that, with a rapid onset of action and improved gastrointestinal tolerability, novel oral potassium binders (e.g., patiromer and sodium zirconium cyclosilicate) are alternative treatment options for both acute and chronic hyperkalaemia. To optimise the care for patients with hyperkalaemia in the Asia–Pacific region, a multidisciplinary expert panel was convened to review published literature, share clinical experiences, and ultimately formulate 25 consensus statements, covering three clinical areas: (i) risk factors of hyperkalaemia and risk stratification in susceptible patients; (ii) prevention of hyperkalaemia for at‐risk individuals; and (iii) correction of hyperkalaemia for at‐risk individuals with cardiorenal disease. These statements were expected to serve as useful guidance in the management of hyperkalaemia for health care providers in the region.
Summary at a glance
To optimise the care for patients with hyperkalaemia in the Asia–Pacific region, a multidisciplinary expert panel was convened to review published literature, share clinical experiences, and ultimately formulate a set of consensus statements.
Type 2 diabetes mellitus (T2DM) has traditionally been considered a coronary heart disease 'risk equivalent' for future mortality, but significant heterogeneity exists across people with T2DM. This ...study aims to determine the risk of all-cause mortality of patients with cardiovascular disease (CVD) and T2DM in UK and Hong Kong, with stratifications for hemoglobin A1 (HbA1c) concentrations, compared with those without CVD and diabetes mellitus.
This is a retrospective cohort study of 3 839 391 adults from Hong Kong and a prospective cohort study of 497 779 adults from the UK Biobank. Individuals were divided into seven disease groups: (1) no T2DM and CVD, (2) T2DM only with HbA1c <7%, (3) T2DM only with HbA1c 7%-7.9%, (4) T2DM only with HbA1c 8%-8.9%, (5) T2DM only with HbA1c ≥9%, (6) CVD only, and (7) T2DM and CVD. Differences in all-cause mortality between groups were examined using Cox regression.
After around 10 years of median follow-up, 423 818 and 19 844 deaths were identified in the Hong Kong cohort and UK Biobank, respectively. Compared with individuals without T2DM and CVD, the adjusted HR for all-cause mortality in the other six disease groups for the Hong Kong cohort was 1.25 (95% CI 1.23 to 1.27) for T2DM only with HbA1c <7%, 1.21 (95% CI 1.19 to 1.23) for T2DM only with HbA1c 7%-7.9%, 1.36 (95% CI 1.33 to 1.39) for T2DM only with HbA1c 8%-8.9%, 1.82 (95% CI 1.78 to 1.85) for T2DM only with HbA1c ≥9%, 1.37 (95% CI 1.36 to 1.38) for CVD only, and 1.83 (95% CI 1.81 to 1.85) for T2DM and CVD, and for the UK Biobank the HR was 1.45 (95% CI 1.33 to 1.58), 1.50 (95% CI 1.32 to 1.70), 1.72 (95% CI 1.43 to 2.08), 2.51 (95% CI 2.05 to 3.08), 1.67 (95% CI 1.59 to 1.75) and 2.62 (95% CI 2.42 to 2.83), respectively. This indicates that patients with T2DM had an increased risk of mortality compared with those without T2DM and CVD, and in those with HbA1c ≥9% an even higher risk than people with CVD.
Patients with T2DM with poor HbA1c control (8%-8.9% and ≥9%) were associated with similar and higher risk of mortality compared with patients with CVD, respectively. Optimal HbA1c, controlled for risk reduction and prevention of mortality and complications in diabetes management, remains important.
Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by ...EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.
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•Engineered ZFA fusions can efficiently target microsatellites across the human genome•EWS-ZFA recapitulates the genome-wide activation of GGAA repeats seen in Ewing sarcoma•KRAB-ZFA silences GGAA repeats and induces selective toxicity in Ewing sarcoma cells•ZFA fusions are tools for functional annotation and therapeutic targeting of repeats
Functionally annotating repetitive sequences in biology and disease remains challenging. Tak et al. show that engineered zinc finger array-based fusions can target microsatellites across the genome and regulate their activities in a cancer model, thereby elucidating insights into genome-wide function of these microsatellites and suggesting potential therapeutic strategies.
This study aimed to evaluate P300 as an electrophysiological marker of cognitive function in patients with systemic lupus erythematosus (SLE) who had previous neuropsychiatric (NPSLE) involvement and ...were diagnosed to have cognitive impairment by standard neuropsychological tests. Event-related potentials (ERPs) were assessed by the auditory and visual oddball paradigms. Amplitude and latency of P300 at the frontal (Fz), central (Cz), and parietal (Pz) regions were determined and compared with controls. P300 detection was performed in NPSLE patients with pre-diagnosed cognitive impairment (n = 9), matched SLE patients without previous NPSLE (non-NPSLE) (n = 9), and healthy controls (n = 15). Auditory oddball task did not show any P300 abnormality between groups. Visual oddball task revealed reduced amplitude of P300 over Fz (P = .002) and Cz (P = .009) electrodes in NPSLE patients compared with healthy controls and among those who had predominant memory deficit (P = .01 at Fz). Abnormal P300 was also observed in non-NPSLE patients at Fz and Cz. Using visual oddball paradigm, abnormal P300 was found in NPSLE patients over frontal and parietal regions compared with normal controls but was not discriminative from possible subclinical disease in non-NPSLE patients. In conclusion, visual oddball paradigm was a more sensitive electrophysiological marker than auditory oddball paradigm for cognitive impairment in NPSLE patients.
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