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Generation of human knock-in organoids by CRISPR-HOT CURRENT

This protocol outlines reagents and procedures to generate human knock-in organoids using our established strategy named CRISPR-HOT. The protocol describes the plasmids to be used and strategies to generate knock-in organoids from different organs.


Introduction
The generation of engineered human adult stem cells (ASC)-derived organoids requires efficient strategies for in vitro genome editing to be applied after the lines have been established.
CRISPR/Cas9 technology has considerably simplified genetic engineering. The used approaches so far were limited to harnessing the homology directed repair (HDR) pathway by taking advantage of a mechanism used by cells to repair double strand breaks (DSBs) which can be introduced at specific sites by 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. Additionally, it requires cloning of the donor plasmid due to the necessity of the presence of homology arms specific to each gene.
Non-homologous end joining (NHEJ), another key DNA repair system, is active in all cell cycle phases, and by ligating DNA ends, does not require regions of homology. Since it is generally believed to be error prone, NHEJ is not widely used for precision transgene insertion. Yet, it has been suggested that NHEJ can be fundamentally accurate and can re-ligate DNA ends without mistakes. Indeed, a handful of studies have exploited NHEJ to ensure the targeted insertion of exogenous DNA in zebrafish, mouse, immortalized human cell lines, and ES cells. We leveraged NHEJ-mediated knock-in to the human organoid field (Artegiani, Hendriks et al.,in press), an approach termed CRISPR-HOT, as a versatile, efficient and robust homology-independent method to obtain knock-in human wild-type organoids from different organs. Here, we describe the protocol of CRISPR-HOT to generate human knock-in organoids.
2) Clone the sgRNA into the empty pSPgRNA (#47108) with the reagents and protocol described in (Ran et al., Nature Protocol 2013).
3) Determine which frame selector to use according to where the sgRNA cuts in the genome (PAM -3nt). For example, if the sgRNA cut leaves a +1 overhang in the ORF, use the frame selector+2, according to (Schmid-Burgk et al. Nature Communications 2016).

Preparation of DNA for transfection
1) Mix together 5 ug of sgRNA plasmid targeting the locus of interest, 5 ug of the appropriate frame selector plasmid (0, 1, or 2), and 5 ug of the universal NHEJ targeting vector of interest.

Transfection of organoids
1) Determine optimal organoid transfection protocol according to the organ. For example, for small intestinal organoids use the protocol as described in (Fujii et al. Nature Protocols 2015). For human liver ductal organoids use the protocol as described in (Artegiani et al. Cell Stem Cell 2019).

Selection of knocked-in organoids
1) When tagging with a fluorescent tag, signal can be readily seen within 3-4 days in the case of endogenously expressed genes. Organoids/cell clumps containing positive cells are picked with a P200 pipette under a fluorescent microscope.
2) Make the single picked organoids into single cells and grow/expand them until a line is generated.
3) Genotype by sanger sequencing of PCR fragments to confirm correct integration. Troubleshooting