Fast and Efficient Knock-Ins was created by‘HOT' CRISPR Tool

Fast and Efficient Knock-Ins was created by‘HOT' CRISPR Tool

The potential that gene-editing tools such as CRISPR-Cas9 hold for eliminating disease and genetic disorders is immense. The ability to excise a mutant gene and replace it with an unaltered version may become the gold standard by which physicians treat patients in the near future. Leading up to these potential new therapies, scientists continue to utilize tools that recapitulate human tissues and body systems as accurately as possible. These mini-organs or organoids have been a major advance for researchers studying various diseases such as cancer. Now, investigators at the Hubrecht Institute in Utrecht have developed a new genetic tool to label specific genes in human organoids.

The novel method, called CRISPR-HOT, was utilized to investigate how hepatocytes divide and how abnormal cells with too much DNA appear. The researchers showed that by disabling the cancer gene TP53, unstructured divisions of abnormal hepatocytes were more frequent, which may contribute to cancer development. Findings from the new study were published recently in Nature Cell Biology through an article titled “Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing.”

Organoids grow from a very small piece of tissue, and researchers have been able to do this for a vast array of organs. The ability to genetically alter these organoids would help a great deal in studying biological processes and modeling diseases. So far, however, the generation of genetically altered human organoids has been proven difficult due to the lack of easy genome engineering methods.

“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,” the authors wrote.

A few years ago, researchers discovered that CRISPR-Cas9, which acts like tiny molecular scissors, can precisely cut at a specific place in the DNA. This new technology greatly helped and simplified genetic engineering.

“The little wound in the DNA can activate two different mechanisms of repair in the cells, that can both be used by researchers to coerce the cells to take up a new part of DNA, at the place of the wound,” remarked study author Delilah Hendriks, PhD, a postdoctoral researcher at the Hubrecht Institute.

One method, called non-homologous end joining, was thought to make frequent mistakes and therefore until now not often used to insert new pieces of DNA. “Since some earlier work in mice indicated that new pieces of DNA can be inserted via non-homologous end joining, we set out to test this in human organoids,” noted lead study investigator Benedetta Artegiani, PhD, a postdoctoral researcher at the Hubrecht Institute.

The research team then discovered that inserting whatever piece of DNA into human organoids through non-homologous end joining is actually more efficient and robust than the other method that has been used until now. They named their new method CRISPR-HOT.

“CRISPR–Cas9-mediated homology-independent organoid transgenesis (CRISPR–HOT), enables efficient generation of knock-in human organoids representing different tissues,” the authors explained. “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 labeled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin—uncovered modes of human hepatocyte division.”

Cell division in 3D organoids shows that healthy (left) organoids show organized division (arrow), 

while organoids in which the cancer gene TP53 is disabled (right) show chaotic cell divisions (arrows). [Benedetta Artegiani, Delilah Hendriks, ©Hubrecht Institute]

The Hubrecht scientists then used CRISPR-HOT to insert fluorescent labels into the DNA of human organoids, in such a way that these fluorescent labels were attached to specific genes they wanted to study. First, the researchers marked specific types of cells that are very rare in the intestine: the enteroendocrine cells. These cells produce hormones to regulate, for example, glucose levels, food intake, and stomach emptying. Because these cells are so rare, they are difficult to study. However, with CRISPR-HOT, the researchers easily “painted” these cells in different colors, after which they easily identified and analyzed them.

Next, the researchers painted organoids derived from a specific cell type in the liver, the biliary ductal cells. Using CRISPR-HOT they visualized keratins, proteins involved in the skeleton of cells. Now that they could look at these keratins in detail and at high resolution, the researchers uncovered their organization in an ultra-structural way. These keratins also change expression when cells specialize or differentiate. Therefore, the researchers anticipate that CRISPR-HOT may be useful to study cell fate and differentiation.

Within the liver, there are many hepatocytes that contain two (or even more) times the DNA of a normal cell. It is unclear how these cells are formed and whether they are able to divide because of this abnormal quantity of DNA. Older adults contain more of these abnormal hepatocytes, but it is unclear if they are related to diseases such as cancer. Artegiani and Hendriks used CRISPR-HOT to label specific components of the cell division machinery in hepatocyte organoids and studied the process of cell division.

“We saw that ‘normal’ hepatocytes divide very orderly, always splitting into two daughter cells in a certain direction,” Artegiani explained.

Hendriks added that “we also found several divisions in which an abnormal hepatocyte was formed. For the first time, we saw how a ‘normal’ hepatocyte turns into an abnormal one.”

In addition to these findings, the researchers studied the effects of a mutation often found in liver cancer, in the gene TP53, on abnormal cell division in hepatocytes. Without TP53 these abnormal hepatocytes were dividing much more often. This may be one of the ways that TP53 contributes to cancer development. The researchers believe that CRISPR-HOT can be applied to many types of human organoids, to visualize any gene or cell type, and to study many developmental and disease-related questions.

story source:geneng news