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The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB−/− and MTTP−/− organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR-Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.

Vector systems based on different retroviruses are widely used to achieve stable integration and expression of transgenes. More recently, transient genetic manipulation systems were developed that are based on integration- or reverse transcription-deficient retroviruses. Lack of viral genome integration is desirable not only for reducing tumorigenic potential but also for applications requiring transient transgene expression such as reprogramming or genome editing. However, all existing transient retroviral vector systems rely on virus-encoded encapsidation sequences for the transfer of heterologous genetic material. We discovered that the transient transgene expression observed in target cells transduced by reverse transcriptase-deficient foamy virus (FV) vectors is the consequence of subgenomic RNA encapsidation into FV particles. Based on this initial observation, we describe here the establishment of FV vectors that enable the efficient transient expression of various transgenes by packaging, transfer, and de novo translation of nonviral RNAs both in vitro and in vivo. Transient transgene expression levels were comparable to integrase-deficient vectors but, unlike the latter, declined to background levels within a few days. Our results show that this new FV vector system provides a useful, novel tool for efficient transient genetic manipulation of target tissues by transfer of nonviral RNAs.