CFTR is a type of ABC (ATP- binding cassette) transporter.CFTR is composed of 2 main transmembrane segments; each of these segments has 6 transmembrane alpha helices for a total of 12 alpha helices.CFTR facilitates the transport of Chloride ions into and out of the cell. The function of the transmembrane transport mechanism depends on the presence of ATP.
First, ATP docks in ATP- binding site on Nucleotide binding domain (NBD). Next, NBD dimerizes and a conformational change occurs in the Transmembrane domains (TMD). The dimerization of the NBDs causes the gate to open. Lastly, ATP is hydrolyzed causing the gate to close.Lgr5 is recognized as a “marker for adult stem cells”Lgr5 is a transmembrane receptor that regulates Wnt/b-catenin and Wnt/PCP signaling pathways. Wnt pathways are used to restore cells and for the growth of cells.The authors used crypts because they can be used as models for making organoids and they are ideal models for this purpose. Ideally, a good model for generating organoids would have a combination of various gastrointestinal cells that can be cultured for extended periods of time.Wnt conditioned media is media containing Wnt- agonistic R-spondin 1. This media was used to identify the murine APC locus of intestinal stem cells in adults.
Because APC is a negative controller of the Wnt pathway, stem cells that have inactive APC alleles, grow when there is no Wnt agonist R-spondin 1 present in the media.N-acetylcysteine (NAC) aids in the protection of intestinal epithelial cells against oxidative stress or damage caused by reactive oxygen species (mainly hydrogen peroxide). The reason NAC is added to the intestinal growth medium, is because NAC helps intestinal tissues to grow by protecting the tissues against oxidative damage. NAC protects tissues from oxidative damage by controlling functions of mitochondria. P38 are a group of mitogen-activated protein kinases (MAPKs); they react to different kinds of stresses exerted on the cell. This group includes four members: p38α, p38β, p38γ, and p38δ. P38 has been shown to be important for normal inflammatory and immune responses in the body.Y-27632 is a Rho kinase inhibitor. It prevents the expansion of cells in media and helps to preserve the cells in media.In cationic lipid mediated transfection, cationic lipids are specifically designed to allow DNA and siRNA to be inserted into cells.
The positive charge on the exterior of the liposome allows it to interact with the surface of the cell membrane with a negative charge. In the procedure, first, the cationic lipid transfection reagent is applied to the liposomes. Then, the cell swallows the liposome via endocytosis to form the DNA-cationic lipid complex. Next, the complex interacts with the cell to affect gene expression.To produce the CFTR targeting vector, first, the human BAC clone was used to PCR amplify the CFTR homology arms. This was done using high-fidelity Phusion Polymerase (NEB). Next, the chosen cassette was also PCR amplified from the pMCS plasmid. The products from the PCR were cloned in pJET1.2 via In-fusion Advanced PCR cloning. Next, silent mutations from the wild type template were added after the PCR. Lastly, the sequences were verified using Sanger Sequencing on a DNA Analyzer. Bacterial artificial Chromosomes (BACs) are plasmids utilized for the purpose of cloning and preserving portions of foreign DNA within E.Coli. BACs can host up to a few hundred kilobases of foreign DNA; they are significant in analyzing genomes of many organisms. In-fusion cloning allows input of DNA products from PCR into almost all plasmids. Using this method of cloning, you can clone any insert wherever you want and into any vector. This method can clone a wide range of DNA portion sizes and can clone several portions at the same time in one reaction. Final products have no extra or unneeded base pairs.
First, choose a vector and identify the site of insertion; then adjust the vector through restriction enzyme digestion or inverse PCR. Next, create PCR primers for the desired gene. Next, amplify the desired gene and ensure it’s the correct gene using agarose gel. Then, use spin column to purify or use cloning enhancer. Then configure, the in-fusion cloning reaction and incubate. Lastly, transform cells with incubated mixture from the In-fusion cloning reaction set up.Purpose:
The purpose of this experiment was to test CRISPR/Cas9 genome editing on intestinal stem cells through targeting the APC locus to see if CRISPR/Cas9 could be used to edit the adult human genome.
In the experiment, CRISPR/Cas9 was used to target APC locus of intestinal stem cells. Because human stem cells (namely intestinal stem cells) need Wnt in the media in order to grow, the author grew stem cells in the medium that did not contain either Wnt or R-spondin in order to see which organoids would grow in the medium.
- In figure 1C, the wild-type intestinal organoids of humans were grown in complete media.
- In figure 1C’, APC mutant intestinal organoids of humans were chosen from medium that did not have Wnt and R-spondin.
- In figure 1D, the desired regions of human APC locus and 5 mutant alleles were chosen from clones.
The results showed that organoids grew exclusively from the medium, with cells that had already been co-transfected with certain sgRNA. The selected clones displayed a cystic morphologic structure; also, genetic sequencing illustrated mutations in the targeted site. This supported that the CRISPR/Cas9 system could be used for editing the genome of intestinal organoids of stem cells in adult humans. In order to repair the the F508del CFTR gene the team looked at the CFTR in primary cultured small and large intestinal cells; they grew organoids of the small and large intestines from two separate patients (who both had the homozygous deletion of phenylalanine at the 508 position). Then they did a forskolin-induced swelling assay which showed that the CFTR did not work properly in the CFTR F508del organoids.They transfected a donor sequence with sgRNAs to cut the CFTR sequence and, then after transfection, used puromycin resistant cells to be tested for integration of the donor vector. Next, they verified the correction of the F508del allele by sequencing the new allele. There was no expression of the repaired allele in the uncorrected control organoids but there was expression in the transgenic clones. RT-PCR was used to confirm knockin events. The F508del allele is the knock out allele.
sgRNA1 and sgRNA2 are used to cut out the F508del allele. The red scissors in the illustration represent the locations where the DNA was cleaved by sgRNAs. The white box on the targeting vector represent the puromycin resistance gene. The silent mutation placed downstream the F508del correction allele permits PCR testing specific to particular alleles. Pp1, Pp2, and Pp3 are PCR primer pairs. Experiment:
This experiment’s main purpose was to demonstrate how CFTR function was redeemed in corrected organoids. To do this, a forskolin assay using transgenic lines was done. Expansion of the organoids was seen using live-cell microscopy (while the control group with untransfected organoids showed no swelling).
The team took intestinal stem cells from adult patients with CF and corrected the F508del allele using CRISPR/Cas9; they were able to show through stretching of organoids surface area that the organoids with corrected alleles functioned.
In transfected organoids, swelling increased by 167% and 177% in for corrected organoid clones from the small intestines; in the large intestines, swelling increased to 187% and 180%. Untransfected organoids barely increased in surface; this correlates with CFTR function due to the F508del allele.