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Biology subjects

Hur, J. K.

Publications and source records attributed to Hur, J. K..

8 recordsLinked to original sources

Fixative eXchange (FX)-seq: Scalable Single-nucleus RNA Sequencing Analysis of PFA-fixed or FFPE Tissue

Single-nucleus RNA sequencing (snRNA-seq) of clinical formalin-fixed, paraffin-embedded (FFPE) samples has long been a challenge due to low reverse transcription (RT) yields. Here, we present Fixative-eXchange (FX)-seq, a highly scalable snRNA-seq method for heavily paraformaldehyde (PFA)-fixed and/or FFPE samples. We employ an organocatalyst to facilitate the removal of PFA crosslinks to increase RT yield and additional regiospecific Pt(II)-based crosslinking of RNA molecules to prevent leakage. FX-seq reveals cellular heterogeneity across multiple fixed samples by analyzing 321,710 nuclei, including PFA-fixed tissue, FFPE blocks, thin FFPE and hematoxylin and eosin (H&E)-stained sections from mouse brain and human cancer specimens such as gastrointestinal stromal tumor and colorectal cancer. FX-seq enables integrated analysis with pathologist annotation to label tumor and non-tumor regions of H&E-stained sections. FX-seq can also be applied to PFA-perfusion-based animal studies, large human cohort studies, and personalized drug treatment through precision medicine.

genomics↗

High-fidelity CRISPR genome editing of single-nucleotide mutation with near-complementary guide RNA via enhanced target binding kinetics

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.

biochemistry↗

shRNAI: a deep neural network for the design of highly potent shRNAs

miRNA-mimicking short hairpin RNA (shRNAmir), which depends on whole miRNA biogenesis, has been used to elucidate the function of target genes and to develop therapeutic approaches due to its stable, robust downregulation. Despite efforts to design potent shRNAmir guide RNAs (gRNAs), biological features other than the sequence have not been fully considered. Here, we developed shRNAI, a convolutional neural network model for the prediction of highly potent shRNAmir gRNAs. The shRNAI model trained with gRNA sequences alone predicted more potent shRNAmir gRNAs than the existing algorithms. The shRNAI+ model, trained with the additional features of shRNAmir processibility and target site context, was further improved on both public and our experimental test datasets. Although the shRNAI models were trained on datasets generated with the CNNC motif-free shRNAmir backbone, they also exhibited better performance for the CNNC motif. Our study provides not only a rational framework for designing shRNAmir gRNAs for targets but also a means of designing optimal RNA interference drugs necessary for RNA therapeutics.

bioinformatics↗

Causal role of the dorsolateral prefrontal cortex in modulating the balance between Pavlovian and instrumental systems in the punishment domain

Previous literature suggests that a balance between Pavlovian and instrumental decision-making systems is critical for optimal decision-making. Pavlovian bias (i.e., approach toward reward-predictive stimuli and avoid punishment-predictive stimuli) often contrasts with the instrumental response. Although recent neuroimaging studies have identified brain regions that may be related to Pavlovian bias, including the dorsolateral prefrontal cortex (dlPFC), it is unclear whether a causal relationship exists. Therefore, we investigated whether upregulation of the dlPFC using transcranial current direct stimulation (tDCS) would reduce Pavlovian bias. In this double-blind study, participants were assigned to the anodal or the sham group; they received stimulation over the right dlPFC for 3 successive days. On the last day, participants performed a reinforcement learning task known as the orthogonalized go/no-go task; this was used to assess each participants degree of Pavlovian bias in reward and punishment domains. We used computational modeling and hierarchical Bayesian analysis to estimate model parameters reflecting latent cognitive processes, including Pavlovian bias, go bias, and choice randomness. Several computational models were compared; the model with separate Pavlovian bias parameters for reward and punishment domains demonstrated the best model fit. When using a behavioral index of Pavlovian bias, the anodal group showed significantly lower Pavlovian bias in the punishment domain, but not in the reward domain, compared with the sham group. In addition, computational modeling showed that Pavlovian bias parameter in the punishment domain was lower in the anodal group than in the sham group, which is consistent with the behavioral findings. The anodal group also showed a lower go bias and choice randomness, compared with the sham group. These findings suggest that anodal tDCS may lead to behavioral suppression or change in Pavlovian bias in the punishment domain, which will help to improve comprehension of the causal neural mechanism. Author summaryA decision-making bias guided by the Pavlovian system (i.e., approach reward and avoid punishment) is often useful and predominant across species but it is also related to several psychiatric conditions. The dorsolateral prefrontal cortex (dlPFC) is known to be related to such "Pavlovian bias" but it is unclear whether a causal relationship exists between them. Here, we evaluated whether decision-making biases including Pavlovian bias could be modulated by exogenous brain stimulation, transcranial current direct stimulation, over the right dlPFC for 3 successive days. A combination of behavioral analysis and computational modeling revealed that the anodal group had lower Pavlovian bias in the punishment domain compared with the sham group. In addition, the anodal group showed lower go bias and choice randomness than the sham group, which can also hamper instrumental learning. These findings suggest a causal role for the dlPFC in modulating the balance between the Pavlovian and instrumental decision-making systems.

neuroscience↗

Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

biochemistry↗

Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

biochemistry↗

Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used to induce double-strand breaks in target DNA and activate the in-vivo DNA repair system for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious and often carcinogenic mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. In this study, we developed a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. We used various genome editors, including CRISPR-Cpf1, Cas9, and an adenine base editor, to induce intracellular genome mutations. The CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6[~]984 fold increase) than did an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow to detect genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.

molecular biology↗