Generating and Utilizing Murine Cas9-Expressing Intestinal Organoids for Large-Scale Knockout Genetic Screening

Abstract

Organoid culture faithfully reproduces in vitro the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumourigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesisbased screening method using murine intestinal organoids by investigating the phenotypical morphology of cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10 days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their un-transduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via Phalloidin and Propidium iodide (PI) staining, proliferation assay and qRT-PCR and also Biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis and drug screening, thereby reducing, and potentially replacing animal models.

Understanding cell‐cell communication and signaling in the colorectal cancer microenvironment

“…47 Organoids serve as excellent in vitro model to study tumor microenvironment, specific cell-type response to drugs and enable cells to grow in a more similar manner to that of living organisms. [79][80][81][82] In a CRC organotypic model, extracellular vesicles from colon fibroblasts grown in hypoxic conditions showed an increase in neoplastic organoids, suggesting a role of fibroblast-derived extracellular vesicles in tumorigenesis 83 . An intestinal organoid consists of multiple epithelial cells.…”

“…Finally, combining single-cell multi-omics data with perturbation experiments such as RNA interference (RNAi) 180 , or CRISPR-Cas9 148 , provides an efficient approach in verifying causal regulatory programs. 81 Positive developments in high-throughput gene technologies such as Perturb-seq combine CRISPR/Cas9-mediated gene perturbation with single-cell sequencing 181 and have been reported to provide the same amount of causal information as RNAi or CRISPR/Cas9 mediated gene activation/deletions while also being less invasive. As singlecell technologies continue to develop, the parameters that can be measured per cell will inevitably increase.…”

CRISPR Screens in Synthetic Lethality and Combinatorial Therapies for Cancer

“…3D cultured organoids recapitulate organ physiological parameters and, as they may be derived from stem cells, these cultures can self-renew and differentiate. Some laboratories have successfully established their own protocols to perform forward genetic CRISPR screens in valuable 3D cultured models: 3D spheroids [ 110 , 111 ] and 3D organoids [ 112 , 113 , 114 , 115 ], despite sgRNA heterogeneous efficiency due to spontaneous intrinsic differentiation or high false positive rates [ 115 ] and challenging genome-wide screen scaling [ 114 ]. These authors support the use of CRISPR screens in 3D cultured cancer models for the identification of gene functions to determine tumorigenesis, disease progression, novel drug targets and reduce or even replace animal models.…”

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

“…Efficient transfection, transgenesis, and clonal isolation methods using 2D hISC cultures would enhance hISC utility for mechanistic studies, disease modeling, and development of high-content platforms for drug screening, however, methods have only been reported for 3D organoids ( Fujii et al., 2015 ; Kashfi et al., 2020 ; Matano et al., 2015 ; Schwank and Clevers, 2016 ). Compared with other methods electroporation resulted in the highest transfection efficiencies in colonoids (approximately 30%) ( Fujii et al., 2015 ), and produced up to 75% efficiencies in other hard to transfect cells cultured as monolayers ( Jordan et al., 2008 ), thus, we hypothesized that electroporation could be used to efficiently transfect hISCs cultured as 2D monolayers.…”