Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Katsuda, Takeshi; Cure, Héctor; Sussman, Jonathan; Simeonov, Kamen P.; Krapp, Christopher; Arany, Zoltàn; Grompe, Markus; Stanger, Ben Z.

doi:10.1002/hep.32759

Cited by 8 publications

(9 citation statements)

References 50 publications

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“…Got2 is the mitochondrial aspartate aminotransferase, with a wide range of substrates and pleiotropic effects, so we chose not to target that enzyme in vivo . We generated these mice using the recently described CRISPR/Cas9-mediated rapid in vivo multiplexed editing (RIME) system 43 (Figure 2E). Briefly, guide RNAs to knockout Kyat1, Aadat, and Kyat3 were generated and integrated into AAV8 vectors for liver-enriched expression.…”

Section: Resultsmentioning

confidence: 99%

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK

Bowman,

Neinast,

Jang

et al. 2024

Preprint

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The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential ″off-target″ effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/-animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/-mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

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Section: Resultsmentioning

confidence: 99%

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK

Bowman,

Neinast,

Jang

et al. 2024

Preprint

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“…1a ). The system enables robust infection of >95% of hepatocytes and, significantly, allows infected cells at different stages of reprogramming to be recognized and isolated by virtue of an EGFP lineage tracer 31 . As a control, mice were injected with AAV8 virus carrying Cre but no additional payload (empty vector; EV).…”

Section: Resultsmentioning

confidence: 99%

“…Mouse HA-Sox4-P2A and P2A-Cre blocks were PCR-amplified from pLVXT-Sox4 (Addgene, #101121) and AAV:ITR-U6-sgRNA(backbone)-TBG-Cre-WPRE-hGHpA-ITR 31 respectively, using the primers listed i n Table S4 . The AAV-TBG backbone was prepared by removing EGFP from pAAV.TBG.PI.eGFP.WPRE.bGH (Addgene, #105535) using NotI-HF (NEB) and BamHI-HF (NEB).…”

Section: Methodsmentioning

confidence: 99%

“…(f) Averaged aggregate plots corresponding to the heatmaps shown in (d). For characterization of the reprogramming stage, 4-5 week-old mice were retro-orbitally injected with AAV:ITR-U6-sgRNA(backbone)-TBG-Cre-WPRE-hGHpA-ITR 31 (empty vector: EV) with 5 × 10 11 genome copies/mouse/gene. One week later, induction of biliary reprogramming was started by initiating a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet (Envigo).…”

Section: Figure Legendsmentioning

confidence: 99%

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Physiological reprogrammingin vivomediated by Sox4 pioneer factor activity

Katsuda

Sussman

Ito

et al. 2023

Preprint

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Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate ″reprogramming″ by opening new chromatin sites for expression that can attract transcription factors from the starting cell′s enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

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“…While CRISPR/Cas9 systems for somatic gene deletion have been described for some tissues, including brain and liver ( 11, 35 ), such a versatile tool has so far been missing for skeletal muscle fibers. Previous work has demonstrated successful somatic gene editing using CRISPR in muscle stem cells (MuSCs) although with rather low efficiency ( 36, 37 ).…”

Section: Discussionmentioning

confidence: 99%

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Thürkauf

Lin

Oliveri

et al. 2023

Preprint

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Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene mutation in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

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Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Cited by 8 publications

References 50 publications

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK

Physiological reprogrammingin vivomediated by Sox4 pioneer factor activity

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

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