Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing

Abstract

rganoids can be generated by guided differentiation of induced pluripotent stem cells and embryonic stem cells, or from cells isolated from adult tissues 1 . Adult stem cell (ASC)-derived organoids are self-organizing structures that recapitulate aspects of cellular composition, three-dimensional (3D) architecture and functionality of the different epithelial tissues from which they originate, while maintaining genomic stability 2,3 . The possibility to derive organoids from genetically modified mouse lines, especially knock-in models, has enabled the generation of engineered mouse organoids that have been used as versatile in vitro tools to answer various biological questions [4][5][6][7][8][9][10] .The generation of engineered human ASC-derived organoids requires that efficient strategies for in vitro genome editing are applied after the lines have been established. CRISPR-Cas9 technology has simplified genetic engineering considerably. To date, these approaches were largely limited to the non-homologous end joining (NHEJ)-mediated introduction of indels into the endogenous loci of organoids, leading to gene mutations [11][12][13][14] . By harnessing the HDR pathway, a single-base substitution was introduced to correct the CFTR locus in cystic fibrosis intestinal organoids 15 , and a few human ASC-organoid knock-in reporter lines have been generated, but mostly in colon cancer organoids [16][17][18] .Knock-in using HDR takes advantage of a mechanism used by cells to repair double-stranded breaks (DSBs). Such breaks can be introduced at specific sites using CRISPR-Cas9. HDR is the most commonly used approach for targeted insertion, but this process is inefficient and requires cells to be in S phase 19,20 . Furthermore, HDR requires that the donor plasmid is cloned, owing to the necessity for the presence of homology arms specific to each gene (Fig. 1a). Recent studies have shown that CRISPR-induced DSBs activate the TP53 damage response and induce a transient cell-cycle arrest in untransformed cells 21 . Permanent or transient inactivation of TP53 increases HDR-mediated knock-in in pluripotent and hematopoietic stem cells 22,23 . Thus, given the demand for novel methods to improve HDR efficiency, inhibition of TP53 was suggested as a potential solution to overcome the low efficiency of HDR-mediated knock-in in untransformed cells 23 .NHEJ, another key DNA repair system, is active in all cell cycle phases 20 and, by ligating DNA ends, does not require regions of homology (Fig. 1a). As NHEJ is generally believed to be error prone, it is not widely used for precision transgene insertion. However, it has been suggested that NHEJ can be fundamentally accurate and can religate DNA ends without errors 24,25 . Indeed, a handful of studies have exploited NHEJ to ensure the targeted insertion of exogenous DNA into zebrafish 26 , mouse 27 , immortalized human cell lines 28,29 and embryonic stem cells 30 . Here we leverage NHEJ-mediated knock-in for use in the human organoid field-an approach named CRISPR-HOT-as a versatile...

Cancer research using organoid technology

“…This observation highlights two considerations when establishing cancer organoid cultures. Firstly, due to higher rates of cell death by mitotic catastrophes and other aberrations [40,88], cancer organoids often grow slower than normal organoids, and, secondly, many normal organoid cultures thrive under surprisingly simple growth conditions or require similar conditions as their cancerous counterparts. To avoid this, different strategies have been developed.…”

“…Comparative mutational analysis of organoid cultures generated from different murine and human tissues further demonstrated that tissue-specific mutational signatures can be defined using organoids [36][37][38]. In addition, organoid cultures are amenable to a wide range of experimental tools, including single-cell transcriptomics [39], gene editing and tagging [40], (whole-mount) imaging [41], xenotransplantation [42], and co-culture with other cells such as immune cells (reviewed in [43]). Another complementary approach is to generate organoids using pluripotent stem cells (PSCs), namely embryonic stem cells or induced pluripotent stem cells (iPSCs) (reviewed in [12,13]).…”

Synthetic Virus-Derived Nanosystems (SVNs) for Delivery and Precision Docking of Large Multifunctional DNA Circuitry in Mammalian Cells

“…The same holds for CRISPR-based approaches and, by slightly changing the design of the donor DNA, NHEJ-based editing has been shown to efficiently produce knock-ins in dividing and non-dividing cells at base pair precision. Furthermore, this strategy, named homology-independent targeted integration (HITI), does not require homology arms and it works in vivo, producing up to 60% of correct editing [18,[46][47][48].…”

Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing

“…To enrich the options to generate reporter cell lines and animal models, the CRISPaint system was developed to introduce reporter tags at the C-terminal coding portion of any gene of interest using a universal donor plasmid and the NHEJ pathway to repair the RGN-induced DSBs (Schmid-Burgk et al, 2016). More recently, this methodology was applied in human organoids, named CRISPR-HOT for homology-independent organoid transgenesis (Artegiani et al, 2020).…”