Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we ...develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.
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•Inducible CRISPRi iPSCs provide a valuable resource for rapid gene knockdown•CRISPRi knockdown is efficient, tunable, and reversible in iPSCs•CRISPRi knockdown is highly specific•CRISPRi enables disease modeling in iPSC-derived cardiomyocytes
In this article, Mandegar and colleagues utilize CRISPR interference for efficient gene knockdown in iPSCs and their differentiated cell derivatives. The CRISPRi tools and cell lines presented in this study are highly versatile and serve as a useful resource for the cell and stem cell biology communities.
Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining ...(NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.
Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed ...an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.
Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: ...homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.
Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated ...missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.
•Children’s ability to follow spoken instructions increases from age 7 to 12 years.•Children were asked to use motor imagery or verbal rehearsal to encode instructions.•Performance was better under ...motor imagery than verbal rehearsal.•Motor imagery benefits were similar for younger and older children.•Motor imagery benefits were similar for verbal and enacted recall.
The ability to follow spoken instructions is critical for children’s learning in school and relies on the storage and processing of information in working memory. This study compared the effects of two encoding strategies (motor imagery and verbal rehearsal) on children’s ability to follow spoken instructions in a working memory paradigm. A total of 146 children aged 7–12 years completed an instruction span task. In this task, children listened to a series of action–object commands and encoded them by either motor imagery or verbal rehearsal. They then attempted to recall the sequence in serial order by either enacted recall or verbal recall. Overall, children’s ability to follow spoken instructions increased with age. In all age groups, children showed superior recall of instructions when they imagined the actions compared with verbal rehearsal of the actions during encoding, and this benefit of motor imagery was similar for verbal recall and enacted recall. Younger children reported motor imagery as more helpful than verbal rehearsal for remembering instructions, whereas older children considered verbal rehearsal as more useful. The study provides novel evidence for motor imagery as a superior strategy (relative to verbal rehearsal) for remembering spoken instructions in school-age children.
Intimate communication between neural and vascular structures is required to match neuronal metabolism to blood flow, a process termed neurovascular coupling. The number of laboratories assessing ...neurovascular coupling in humans is increasing due to clinical interest in disease states, and basic science interest in a non-anesthetized, non-craniotomized, unrestrained, in vivo model. However, there is a lack of knowledge regarding how best to characterize the neurovascular response. To address this knowledge gap, we have amassed a highly powered human neurovascular coupling dataset, and deployed a network-based approach to reveal the most powerful and consistent metrics for quantifying neurovascular coupling. Using dimensionality reduction, community-based clustering, and majority-voting of traditional metrics (e.g. peak response, time to peak) and non-traditional metrics (e.g. varying time windows, pulsatility), we have identified which of the existing metrics predominantly characterize the neurovascular coupling response, are stable within and across participants, and explain the vast majority of the variance within our dataset of over 300 trials. We then harnessed our empirical approach to generate powerful novel metrics of neurovascular coupling, termed iAmplitude, iRate, and iPulsatility, which increase sensitivity when capturing population differences. These metrics may be useful to optimally understand neurovascular coupling in health and disease.
The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide ...substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.
This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis ...probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them.
The role of endoscopic procedures, in both diagnostic and therapeutic purposes is continually expanding and evolving rapidly. In this context, endoscopists will encounter patients prescribed on ...anticoagulant and antiplatelet medications frequently. This poses an increased risk of intraprocedural and delayed gastrointestinal bleeding. Thus, there is now greater importance on optimal pre, peri and post-operative management of anticoagulant and/or antiplatelet therapy to minimise the risk of post-procedural bleeding, without increasing the risk of a thromboembolic event as a consequence of therapy interruption. Currently, there are position statements and guidelines from the major gastroenterology societies. These are available to assist endoscopists with an evidenced-based systematic approach to anticoagulant and/or antiplatelet management in endoscopic procedures, to ensure optimal patient safety. However, since the publication of these guidelines, there is emerging evidence not previously considered in the recommendations that may warrant changes to our current clinical practices. Most notably and divergent from current position statements, is a growing concern regarding the use of heparin bridging therapy during warfarin cessation and its associated risk of increased bleeding, suggestive that this practice should be avoided. In addition, there is emerging evidence that anticoagulant and/or antiplatelet therapy may be safe to be continued in cold snare polypectomy for small polyps (< 10 mm).
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