Expanding RNA editing toolkit using an IDR-based strategy.

RNA base editors should ideally be free of immunogenicity, compact, efficient, and specific, which has not been achieved for C > U editing. Here we first describe a compact C > U editor entirely of human origin, created by fusing the human C > U editing enzyme RESCUE-S to Cas inspired RNA targeting system (CIRTS), a tiny, human-originated programmable RNA-binding domain. This editor, CIRTS-RESCUEv1 (V1), was inefficient. Remarkably, a short histidine-rich domain (HRD), which is derived from the internal disordered region (IDR) in the human CYCT1, a protein capable of liquid-liquid phase separation (LLPS), enhanced V1 editing at on-targets as well as off-targets, the latter effect being minor. The V1-HRD fusion protein formed puncta characteristic of LLPS, and various other IDRs (but not an LLPS-impaired mutant) could replace HRD to effectively induce puncta and potentiate V1, suggesting that the diverse domains acted via a common, LLPS-based mechanism. Importantly, the HRD fusion strategy was applicable to various other types of C > U RNA editors. Our study expands the RNA editing toolbox and showcases a general method for stimulating C > U RNA base editors.

Large-scale multiplexed mosaic CRISPR perturbation in the whole organism.

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.

IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2-specific CAR T cells targeting breast cancer cells did not affect CAR expression or CAR T cell differentiation, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model, which was associated with increased T cell activation and proliferation. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.

Programmable C-to-U RNA editing using the human APOBEC3A deaminase.

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.

In situ conversion of defective Treg into SuperTreg cells to treat advanced IPEX-like disorders in mice.

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity,  with  Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.

Effect of Third-generation Dual-source CT Technology on Image Quality of Low-dose Chest CT.

Objective To evaluate the image quality and radiation dose of third-generation dual-source CT with tin filtration for spectral shaping and iterative reconstructions.Methods Thirty-five patients underwent low-dose CT (LDCT) for lung cancer screening on second-generation dual-source CT and follow-ups on third-generation dual-source CT. Image quality and radiation dose were compared between the two examinations.ResultsThe radiation dose of third-generation dual-source CT [dose-length product (DLP)(49.7±18.2)mGy·cm, effective dose (ED)(0.73±0.26)mSv] was lower than second-generation dual-source CT [DLP (86.37±13.44) mGy·cm, ED(1.20±0.42)mSv](t=6.01, P=0.000;t=6.57, P=0.000). The objective image noise of second-generation dual-source CT [(25.7±2.9)HU] was higher than that of third-generation dual-soure CT[(18.6±4.2)HU](t=5.24,P=0.000).The subjective image noise of second-generation dual-source CT [(4.60±0.49)scores] was significantly lower than that of third-generation dual-source CT [(4.80±0.40)scores] (t=4.15, P=0.000). Conclusion Chest CT for the detection of pulmonary nodules can be performed with third-generation dual-source CT that produces high image quality and low radiation dose when using a stellar infinity detector with spectral shaping.

Value of CT Density Measurements in Distinguishing Parathyroid Adenoma,Thyroid,and Lymph Node.

Objective To explore the value of multi-phase contrast-enhanced computed tomography in the differential diagnosis of parathyroid adenoma,lymph node,and thyroid. Methods The enhanced multi-slice CT (MSCT) results of 21 parathyroid adenoma patients were analyzed,and their postoperative pathological specimens were examined. During the MSCT,the plain CT scan was recorded,along with the density of thyroid adenoma,lymph nodes,and thyroid at 35 s and 65 s (D0,D35,D65) following the injection of contrast medium. Results During the D0 phase,there was significant difference in CT values between the parathyroid adenoma and thyroid parenchyma[(45?12) HU vs.(90?15)HU,P=0.007]. According to ROC curve,75 HU,with 95.2% sensitivity and specificity,was the critical value for distinguishing the density of parathyroid adenoma and that of thyroid parenchyma. At 35 s following the injection of contrast medium,there was significant difference in the enhancement degree between parathyroid adenoma and lymph node[(182?39) HU vs.(80?20)HU,P=0.004]. According to ROC curve,111 HU,with 95.2 % sensitivity and specificity,was the critical value for distinguishing the density of parathyroid adenoma and that of lymph node 35 s following the injection of contrast medium. At 35 s to 65 s following the injection of contrast medium,the parathyroid adenoma experienced a decline in density,which was dramatically different from parathyroid adenoma,however,lymph node experienced a rise in density. Conclusion Enhanced CT measurements at different time points enable the differentiation among parathyroid adenomas,lymph nodes,and thyroid.

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging for Monitoring the Instantly Therapeutic Efficacy of Radiofrequency Ablation in Rabbit VX2 Tumors without Evident Links between Conventional Perfusion Weighted Images.

OBJECTIVE: To investigate the intravoxel incoherent motion diffusion weighted imaging (IVIM-DWI) as a potential valuable marker to monitor the therapy responses of VX2 to radiofrequency ablation (RF Ablation). METHODS: The institutional animal care and use committee approved this study. In 10 VX2 tumor-bearing rabbits, IVIM-DWI examinations were performed with a 3.0T imaging unit by using 16 b values from 0 to 800 sec/mm2. The true diffusion coefficient (D), pseudodiffusion coefficient (D*) and perfusion fraction (f) of tumors were compared between before and instantly after RF Ablation treatment. The differences of D, D* and f and conventional perfusion parameters (from perfusion CT and dynamic enhanced magnetic resonance imaging, DCE-MRI) in the coagulation necrosis area, residual unablated area, untreated area, and normal control had been calculated by compared t-test. The correlation between f or D* with perfusion weighted CT including blood flow, BF (milliliter per 100 mL/min), blood volume, BV (milliliter per 100 mL/min), and capillary permeability-surface area, PMB (as a fraction) or from DCE-MRI: transfer constant (Ktrans), extra-vascular extra-cellular volume fraction (Ve) and reflux constant (Kep) values had been analyzed by region-of-interest (ROI) methods to calculate Pearson's correlation coefficients. RESULTS: In the ablated necrosis areas, f and D* significantly decreased and D significantly increased, compared with residual unblazed areas or untreated control groups and normal control groups (P < 0.001). The IVIM-DWI derived f parameters showed significant increases in the residual unablated tumor area. There was no significant correlations between f or D* and conventional perfusion parameters. CONCLUSIONS: The IVIM-DW derived f, D and D* parameters have the potential to indicate therapy response immediately after RF Ablation treatment, while no significant correlations with classical tumor perfusion metrics were derived from DCE-MRI and perfusion-CT measurements.

Thermochemical ablation therapy of VX2 tumor using a permeable oil-packed liquid alkali metal.

OBJECTIVE: Alkali metal appears to be a promising tool in thermochemical ablation, but, it requires additional data on safety is required. The objective of this study was to explore the effectiveness of permeable oil-packed liquid alkali metal in the thermochemical ablation of tumors. METHODS: Permeable oil-packed sodium-potassium (NaK) was prepared using ultrasonic mixing of different ratios of metal to oil. The thermal effect of the mixture during ablation of muscle tissue ex vivo was evaluated using the Fluke Ti400 Thermal Imager. The thermochemical effect of the NaK-oil mixture on VX2 tumors was evaluated by performing perfusion CT scans both before and after treatment in 10 VX2 rabbit model tumors. VX2 tumors were harvested from two rabbits immediately after treatment to assess their viability using trypan blue and hematoxylin and eosin (H.E.) staining. RESULTS: The injection of the NaK-oil mixture resulted in significantly higher heat in the ablation areas. The permeable oil controlled the rate of heat released during the NaK reaction with water in the living tissue. Perfusion computed tomography and its parameter map confirmed that the NaK-oil mixture had curative effects on VX2 tumors. Both trypan blue and H.E. staining showed partial necrosis of the VX2 tumors. CONCLUSIONS: The NaK-oil mixture may be used successfully to ablate tumor tissue in vivo. With reference to the controlled thermal and chemical lethal injury to tumors, using a liquid alkali in ablation is potentially an effective and safe method to treat malignant tumors.