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"Holden, Kevin"
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Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression
Background
Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the
Tspan2
promoter.
Results
Quantitative RT-PCR showed loss of
Tspan2
mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the
Tspan2
CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the
Tspan2
CArG box displayed similar cell-specific loss of
Tspan2
mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of
Tspan2
in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.
Conclusions
PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
Journal Article
Enhanced RNA-targeting CRISPR-Cas technology in zebrafish
CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.
CRISPR-Cas13 RNA-targeting systems comprise an invaluable set of tools in the fields of basic and applied sciences. Here, Moreno-Sánchez, Hernández-Huertas, and Nahón-Cano et al. enhanced the use of the CRISPR-RfxCas13d system in zebrafish for targeted depletion of endogenous mRNAs.
Journal Article
Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here the authors show that CRISPR-Cas13d can be used as a broad-spectrum antiviral to inhibit human coronaviruses, including new SARS-CoV-2 variants, combined with small molecule drugs for an enhanced antiviral effect in human primary cells.
Journal Article
Exploring the frontiers of science in Western China
Abstract only China's remarkable economic rise is also propelling advances across the sciences, and these intertwined trends are transforming the western section of the country. To speed up this transformation, the Chinese Academy of Sciences (CAS), universities, and the central government are all creating award schemes aimed at attracting leading scholars trained abroad to take up positions in western China. Science centers across the region are ramping up efforts to make research teams more innovative and more international. This strategy extends to the spheres of genomics, medicine, geoscience, astronomy, physics, and conservation and is creating professional openings across the western sector of China.
Journal Article
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Large sequences are integrated site specifically into the human genome without double-strand DNA cleavage.
Journal Article
In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels
Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting
PCSK9
, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.
Base editors are effective and safe for cholesterol reduction in non-human primates.
Journal Article
BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2
SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for
ACE2
transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous
ACE2
expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19.
Samelson et al. report that BRD2 inhibition suppresses SARS-CoV-2 infection in epithelial cells and Syrian hamsters via reducing the transcription of host cell surface receptor ACE2.
Journal Article
Generation of precision preclinical cancer models using regulated in vivo base editing
Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.
The function of single-nucleotide variants can now be tested in a base editor mouse model.
Journal Article