生物醫學導讀(二)
CRISPR-HOT: A new tool to 'color' specific genes and cells
10.03.20
A Brief Introduction to the Article
CRISPR-HOT: A new tool to 'color' specific genes and cells
Published by ScienceDaily in 2nd March 2020
https://www.sciencedaily.com/releases/2020/03/200302113330.htm
408257009, Chieh-Lun Kan, BMS 1, CCU
FULL STORY
Researchers from the group of Hans Clevers at the Hubrecht Institute have developed
a new genetic tool to label specific genes in human organoids, or mini organs. They
used this new method, called CRISPR-HOT, to investigate how hepatocytes divide
and how abnormal cells with too much DNA appear. By disabling the cancer gene
TP53, they showed that unstructured divisions of abnormal hepatocytes were more
frequent, which may contribute to cancer development. Their results were described
and published in the scientific journal Nature Cell Biology.
Organoids are mini organs that can be grown in the lab. These mini-organs grow from
a very small piece of tissue, and this is possible for various organs. The ability to
genetically altering these organoids would help a great deal in studying biological
processes and modelling 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-HOT
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" says Delilah Hendriks (Hubrecht Institute). One of these methods, 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" says Benedetta Artegiani
(Hubrecht Institute). Artegiani and Hendriks 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.
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生物醫學導讀(二)
CRISPR-HOT: A new tool to 'color' specific genes and cells
10.03.20
Coloring Cells
The researchers 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 CRISPRHOT, the researchers easily "painted" these cells in different colors, after which they
easily identified and analyzed them. Second, the researchers painted organoids
derived from a specific cell type in the liver, the biliary ductal cells. Using CRISPRHOT 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.
Abnormal Cell Division in The Liver
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. Artegiani: "We saw that "normal" hepatocytes divide very orderly, always
splitting into two daughter cells in a certain direction." Hendriks: "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 this, 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.
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生物醫學導讀(二)
CRISPR-HOT: A new tool to 'color' specific genes and cells
10.03.20
JOURNAL REFERENCE
1. Benedetta Artegiani, Delilah Hendriks, Joep Beumer, Rutger Kok, Xuan Zheng, Indi
Joore, Susana Chuva de Sousa Lopes, Jeroen van Zon, Sander Tans, Hans
Clevers. Fast and efficient generation of knock-in human organoids using
homology-independent CRISPR–Cas9 precision genome editing. Nature Cell
Biology, 2020; DOI: 10.1038/s41556-020-0472-5
KEYWORDS AND TECHINICAL TERMS
Organoid or mini organ is a miniature model of real organ made from its tissues or
stems cells, created for easier manipulation and observation in vitro.
CRISPR-HOT is the abbreviation for CRISPR–Cas9-mediated homologyindependent organoid transgenesis and it’s the highlight of the article. In the series of
experiments by Artegiani, Hendriks et al. used this way to “colour” cells of interest.
Hepatocyte in liver is in charge of basic functions of liver (synthesis of cholesterol
and proteins, etc.) and makes up 55 – 65% of liver’s mass.
TP53 is the gene responsible for the production of tumour suppressing protein p53. In
this article, the researchers found that, undoubtedly, without this gene hepatocytes
were found dividing abnormally.
Non-homologous end joining (NHEJ) is a mean of DNA repair without the presence
of its homologous counterpart as a template. When the overhanging regions of both
ends have perfect compatibility, then the repair will be precise. Sadly, overhangs, in
most cases, do not match in such an ideal way, thus, making NHEJ less reliable.
Enteroendocrine cell found in gastrointestinal tract releases hormones in response to
stimuli so that the condition can be optimised.
Biliary ductal cell. I’m not really sure about this one. The term “biliary duct” used in
the original report is ambiguous and I can barely find a piece of information on the
Internet. Also, the cited paper requires a fee as little as 8.99 USD to be unlocked. I
couldn’t afford it as a student.
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生物醫學導讀(二)
CRISPR-HOT: A new tool to 'color' specific genes and cells
10.03.20
BRIEFING
Two major findings can be inferred from the article:
1. The Potential of the Novel Technology, CRISPR-HOT.
In the past, genetical alteration of organoids is difficult to achieve due to a lack of a
proper method. However, CRISPR-HOT, which is derived from NHEJ, made it
possible.
In this case, CRISPR-HOT is used to colour the selected genes by attaching
fluorescent genes to them. Several applications of this feature are demonstrated, also.
The first test subject to be highlighted is enteroendocrine cell, which is uncommon to
be found. Then, keratin in biliary ductal cells that is flexible in shape is the next, and
it was proven changing expression when the cell is differentiating.
These results are made possible by the introduction of CRISPR-HOT and the scholars
are looking forward to expand its usage to other organoids to visualize the complex
structure of them.
2. With the Absence of TP53, Occurrence of Abnormal Division of Hepatocytes
is more Common.
Additional experiment with help of CRISPR-HOT points out that hepatocytes without
TP53 is more easily to divide disorderly. That indicates one of possible relation
between TP53 and tumours. Also, the older the patient is, the more likely they’re to
carry abnormal hepatocytes.
PERSONAL OPINION
I have never heard of organoid before, so my knowledge of it may be incorrect.
Anyway, in the first time I heard of it, the concept using it in the field of neuroscience
occurs to me. If a miniature liver can be made use of, then it will be easier to study
interaction between neurons with a miniature brain. Then I found a recent report in
which they said it will be “inaccurate” and the report itself devastated my ideas
(details in Section 3 of Discussion).
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生物醫學導讀(二)
CRISPR-HOT: A new tool to 'color' specific genes and cells
10.03.20
As for our main focus, CRISPR-HOT, its mechanism was hardly interpreted in the
article. But I am amazed, still, by the application of it in man-made organoids. I can
think of a few other usages of it, no matter possible or not. The technology is still in
the phase of growing, so we can be optimistic about its potential. On the contrary, its
risk is not mentioned. The off-target effect of CRISPR/Cas9 is notorious, and the
ethical questions behind it is even more notorious. It’s likely that its spin-off,
CRISPR-HOT, will be exposed in the same risk. The pros and cons of using it is yet to
be evaluated.
DISCUSSION
1.
2.
3.
NHEJ is inaccurate in regular cells, but is precise in organoid, why?
Perhaps living organs are much more complex so it can’t be manipulated easily.
What is the possible mechanism that can explain that “without TP53, a cell
is more likely to grow into a tumour”?
TP53 synthesize a gene repairing protein called P53, thus, without the former,
the latter will not prevail. Consequently, defected DNA are expected not to be
repaired, making the cell the expression of oncogenes to be more common to
occur.
In what field else can CRISPR-HOT be used?
In a recent report (Not ‘Brains in a Dish’: Cerebral Organoids Flunk Comparison
to Developing Nervous System on UCSF, 29th Jan., 2020), scholars in UCSF
doubted that the widely used cerebral organoid will precisely replicate the
function of a human brain, because differentiation in vitro is not as the same as
that is in vivo. Thus, I think that this method may be full of potential to induce
the specialization of brain cells.
4.
5.
It can be a good to for studies of morphogenesis, too!
Is it possible to enhance it so we can use it in vivo?
Personally, I think it’s not likely to happen, especially in human. Altering gene
sequence of cells in a living animal is much harder than in a petri dish. We are
not sure about where will these proteins, include CRISPR/Cas9 system, Ku
proteins, DNA-PKcs, etc., operate. Possibility of unwanted gene change too high
to be ignored.
If we can use it in vivo, will it be better than in situ hybridization in locating
genes?
I think in situ hybridization is much simpler in mechanism and cost less.
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