The latest research progress of CRISPR/Cas in 2022

The genome editing technology CRISPR/Cas9 was listed by Science magazine as one of the top ten scientific and technological advances of the year in 2013, and was highly valued
by people.
In October 2020, Dr.
Emmanuelle Charpentier of the Max Planck Institute for Pathogenology in Germany and Dr.
Jennifer A.
Doudna of the University of California, Berkeley were awarded the 2020 Nobel Prize
in Chemistry for their contributions to CRISPR-Cas9 gene editing.

CRISPR is short for regular spaced clustered short palindromic repeats, and Cas is short
for CRISPR-related proteins.
Originally discovered in bacteria, CRISPR/Cas is a defense system
used by bacteria to recognize and destroy invasion by bacteriophages and other pathogens.

On November 26, 2018, Chinese scientist He Jiankui claimed that the world's first gene-edited babies--- twin female babies--- were born
in November.
He used a powerful gene-editing tool, CRISPR-Cas9, to modify one of the twins' genes so that they were born naturally resistant to HIV infection
.
This is also the world's first case of immune AIDS gene-edited babies
.
This news instantly fermented rapidly on domestic and foreign websites, causing thousands of waves
.
Some scientists support He Jiankui's research, but more question, even condemn.

The editor has combed through the recent news on CRISPR/Cas research reported by Biovalley for everyone to read
.

1.
Two Science reveals that RNA-guided cleavage of CRISPR-Cas effector proteins triggers adaptive immune responses

doi:10.
1126/science.
add7450; doi:10.
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In the first new study, researchers from the Massachusetts Institute of Technology demonstrated that the CRISPR-associated protease Csx29 exhibits programmable RNA-activated endopeptidase activity against factor σ inhibitors to regulate transcriptional responses
.
The cryo-EM structure of the active substrate-bound Csx29 complex reveals a heterogeneous activation mechanism that recombines catalytic residues of Csx29 upon target RNA binding
.
The findings were published in the Nov.
26, 2022 issue of Science in the paper "RNA-activated protein cleavage with a CRISPR-associated endopeptidase.
"
This discovery reveals an RNA-guided function in nature that can be used for RNA detection applications
in vitro and in human cells.

In a second new study, researchers from the Massachusetts Institute of Technology in the United States and the University of Tokyo in Japan found that the type III-E CRISPR system includes the proteinease Csx29, similar to caspase, which is activated to cleave another helper protein
called Csx30 when Cas7-11 recognizes the target RNA.
This cleavage produces toxic Csx30 fragments that are hypothesized to inhibit specialized σ factor RpoE, regulate the bacterial response to phage infection, and cause bacterial growth arrest, thereby clearing phage infection
。 The findings were published in the Nov.
26, 2022 issue of Science in the paper "RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.
"

They also reported the cryo-EM structure of the Cas7-11-crRNA-Csx29 complex in the presence and absence of target RNA, and demonstrated that binding of target RNA induces conformational changes
in Csx29.
Biochemical experiments have shown that cleavage of Csx29 to the helper protein Csx30 is target RNA-dependent
.
The recombination of this system in bacteria shows that cleavage of Csx30 produces toxic protein fragments that lead to growth arrest, which is regulated by Csx31
.
Csx30 binds to Csx31 and the related σ factor RpoE, suggesting that Csx30-mediated σ factor RpoE inhibition regulates bacterial cell response
to phage infection.
We program the Cas7-11-CSX29-CSX30 system for programmable RNA detection
in mammalian cells.
These findings expand the known complexity of CRISPR immune responses, enabling protease-based programmable RNA detection in mammalian cells
.
Overall, the Cas7-11-Csx29 effector protein is an RNA-dependent nuclease-protease
.

2.
Cell: The discovery of the CRISPR-Cas system in thousands of viruses holds promise for improving cell genome editing

doi:10.
1016/j.
cell.
2022.
10.
020

Systematic scanning of viral genomes reveals a plethora of potential CRISPR-based genome editing tools
.
The CRISPR-Cas system is common in the microbial world of bacteria and archaea that often help their host cells fight off viruses
.
But in a new study, researchers from the University of California, Los Angeles found that the CRISPR-Cas system accounts for 0.
4 percent
of the genome sequences of viruses known as bacteriophages that can infect these microbes.
They believe that the viruses use CRISPR-Cas to compete with each other--- and may also be manipulating the genetic activity of the host to their advantage
.
The results were published in the November 23, 2022 issue of Cell in the paper "Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors
.
"

Viruses sometimes acquire fragments of their host's genome, and scientists have previously found CRISPR-Cas
in viral genomes.
If these stolen DNA fragments give the virus a competitive advantage, they can be preserved and gradually modified to better serve
the virus's lifestyle.
For example, a virus infected with Vibrio cholerae uses CRISPR-Cas to cut and inactivate
the DNA that codes for antiviral defenses in bacteria.

In the new study, Jennifer Doudna, a molecular biologist at the University of California, Berkeley, and Jillian Banfield, a microbiologist, and colleagues decided to look more holistically for the CRISPR-Cas system
in viruses that infect bacteria and archaea (i.
e.
, bacteriophages).
To their surprise, they found about 6,000 bacteriophages encoding the CRISPR-Cas system, including representatives of
every known type of CRISPR-Cas system.
Doudna said, "There is evidence that these CRISPR-Cas systems are useful for bacteriophages
.
”

The authors found widespread variation in the usual CRISPR-Cas structure, with some CRISPR-Cas systems missing components and others unusually compact
.
Anne Chevallereau, who studies phage ecology and evolution at the French National Center for Scientific Research, said, "Even though phage-encoded CRISPR-Cas systems are rare, they are highly diverse and widely distributed
.
Nature is full of surprises
.
”

3.
Science: Phase 1 clinical trials have shown that universal CAR-T cells edited by CRISPR genes can be used to treat recurrent B-cell leukemia

doi:10.
1126/scitranslmed.
abq3010

In a new clinical study, researchers from Great Ormond Street Hospital for Children and University College London in the UK used CRISPR/Cas9 technology to genetically modify donor T cells in an attempt to treat severely ill children with drug-resistant leukemia who have exhausted all available treatments
.
The Phase I clinical trial is the first to use "universal" CRISPR-edited T cells in humans and represents an important step
forward in using gene-edited cells to treat cancer.
As part of this clinical trial, they built and applied a new generation of more precise "universal" genome-edited T cells
.
The results were published in the October 26, 2022 issue of Science Translational Medicine in the paper "Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia"
。

Image from Science Translational Medicine, 2022, doi:10.
1126/scitranslmed.
abq3010
.

The authors used CRISPR to genetically modify T cells, specifically by cutting the T cell's DNA and inserting a piece of genetic code
.
In this case, this genetic code allows T cells to express a chimeric antigen receptor (CAR) that recognizes a marker called CD19 on the surface of cancerous B cells, and the resulting CD19 CAR-T cells destroy these cancerous B cells
.
They then used CRISPR to destroy the T cell receptor α strands in CD19 CAR-T cells and remove their CD52, thereby obtaining TT52CAR19 T cells, thus constructing a universal CAR-T cell therapy that can be used "off-the-shelf" without any donor matching
.

While the NHS now offers some CAR T-cell therapies, they rely on collecting patients' own T cells and genetically modifying
them.
This is expensive, not always feasible, and impossible to achieve
in a short period of time.
Genome editing is currently being studied to allow donor-supplied T cells to be pre-fabricated for use in multiple patients, with the aim of reducing costs and making them more accessible
.

Nat Biotechnol: Utilizes an improved CRISPR-Cas9 gene-editing system to efficiently introduce longer DNA sequences into precise sites in the cell's genome

doi:10.
1038/s41587-022-01418-8

A new variant of the CRISPR-Cas9 gene-editing system makes it easier to genetically engineer
large numbers of cells for therapeutic applications.
The method, developed by the Glassstone Institute and the University of California, San Francisco, allows scientists to introduce exceptionally long DNA sequences into precise locations in a cell's genome with very high efficiency, without the need for the viral delivery systems
traditionally used to carry DNA into cells 。 The results of the study were published online on August 25, 2022 in the journal Nature Biotechnology as "High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.
"

DNA can exist in single-stranded or double-stranded form, to which Cas9 attaches to double-stranded DNA
.
The authors soon discovered that high levels of double-stranded DNA templates were toxic to cells, so the method could only be used with low amounts of template DNA, which led to inefficient editing
.

Marson's team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations
.
So, in this new paper, they describe a way to attach the modified Cas9 enzyme to a single-stranded DNA template by simply adding a small dangling double-stranded DNA
at either end.
"It gives us a balanced, best of both worlds
," Marson said.
”

Comparison of CTS (Cas9 target sequence) template design, image from Nature Biotechnology, 2022, doi:10.
1038/s41587-022-01418-8
.

Single-stranded DNA templates can more than
double the efficiency of gene editing compared to traditional double-stranded DNA templates.
The double-stranded ends of single-stranded DNA molecules allow people to use Cas9 to enhance the delivery
of nonviral vectors in cells.

In the new study, the authors used the new DNA template to generate more than 1 billion CAR-T cells
targeting multiple myeloma.
CAR-T cells are genetically engineered T cells that can effectively fight specific cells or cancer
.
With the new Cas9-directed single-stranded template, about half of the T cells acquired the new gene and were thus transformed into CAR-T cells
.

Co-author Dr.
Justin Eyquem, assistant professor of medicine in the Department of Hematology and Oncology at UCSF, said, "We know that targeting DNA templates to specific sites in the genome called TRAC (T-cell receptor α α constant) sites will improve the anti-tumor potency
of CAR-T cells.
This new non-viral approach allows us to achieve this more efficiently, which will accelerate the development of
next-generation CAR-T cell therapies.
" ”

5.
Science: Significant progress! Revealing the mechanism of action of CRISPR RNA-guided proteases is expected to provide new antiviral tools and tissue engineering tools

doi:10.
1126/science.
add5064

In a new study, researchers from Cornell University in the United States, Delft University of Technology in the Netherlands, and Pohang University of Science and Technology in South Korea provide new insights into a range of CRISPR systems that could lead to promising antiviral tools and tissue engineering tools
in animals and plants.
They focused on the newly discovered CRISPR RNA-guided Caspase (CRISPR RNA-guided Caspase, Craspase) system
.
The results of the study were published online in Science on August 25, 2022, under the paper "Craspase is a CRISPR RNA-guided, RNA-activated protease"
.
The corresponding authors are Dr.
Ailong Ke, Professor of Molecular Biology and Genetics in Cornell University's College of Arts and Sciences, and Stan J.
J.
Brouns, Ph.
D.
, of Delft University of Technology.

Image from Science, 2022, doi:10.
1126/science.
add5064
.

The CRISPR-Cas system is an RNA-guided nuclease in bacteria that cleaves viral DNA or RNA targets at precise locations for powerful genome editing applications
.
Cysteine aspartate proteases (Caspase) are a family of proteases that control programmed cell death
in animals, including humans.
A recent discovery that proteins similar to Caspase can be linked to CRISPR-Cas have excited the scientific community
.
This CRISPR-guided caspase was given a new name, Craspase
.

In this paper, the authors used cryo-EM maps against the Cracspa system to explain how they cut target RNAs and activate proteases
that can break down proteins.
Ke said, "These cryo-EM maps led to a high-definition molecular film
.
By looking back and forth, we know exactly how Craspa recognizes the RNA target, how this activates the protease, how long this activity lasts, and what ultimately shuts down the activity
of this protease.
Ideas on how to draw power from this platform started pouring in
.
”

6.
Nat Commun: Watch out! CRISPR/Cas9 gene editing may lead to cytotoxicity and genomic instability

doi:10.
1038/s41467-022-32285-1

CRISPR/Cas9, commonly known as "gene scissors," is a precise gene editing technique
.
It allows the desired DNA sequence to be introduced to (almost) anywhere in the genome, modifying or inactivating a gene
.
The technology is widely used in biomedical research, and several CRISPR/Cas9-based therapies are in clinical trials to treat human blood disorders, certain types of cancer and HIV infection, among other diseases
.

In a new study, Dr.
Fran Supek, a researcher at the Institute of Biomedical Research in Barcelona, Spain, and his research team report that CRISPR/Cas9 gene editing can cause cytotoxicity and genomic instability
depending on the target sequence site of the human genome.
This unwanted effect is mediated by the key tumor suppressor protein p53 and is determined
by DNA sequences near the editing site and multiple epigenetic factors in the surrounding region 。 The results of the study were published in the journal Nature Communications on August 4, 2022, with the paper "TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening"
.

TP53 wild-type background may confound estimates of gene selection in genetic screening, image from Nature Communications, 2022, doi:10.
1038/s41467-022-32285-1
.

Using computational methods, the authors analyzed the most popular CRISPR libraries designed for human cells and detected 3300 target sequence sites that showed strong toxic side effects
.
They also report that about 15 percent of human genes contain at least one editing site that shows toxic side effects
.

Dr.
Supek explains, "Our study addresses an important issue with the toxic side effects of Cas9 associated with TP53--- and has been controversial recently, and it also provides guidance
on how to circumvent this issue.
" Avoiding editing at these 'risk' sites would not only make CRISPR editing more effective, but more importantly safer
.
" ”

7.
Nature: Using CRISPR to add reverse transcript barcodes to record the expression order of genes in DNA

doi:10.
1038/s41586-022-04994-6

If you want to track a person's activity throughout the day, you can call them every ten minutes and ask what they're doing
.
An easier approach, though, is to provide them with a diary to record their actions
.
Scientists often rely on a method similar to the first to track how cells change over time; They picked cells from a group of cells at a set time point and took a snapshot
of their gene activity.

Now, in a new study, researchers from the Gladstone Institute in the United States have developed a tool more like a diary or receipt book--- which can record a cell's gene activity for days
at a time.
The tool, called Retro-Cascorder, records data within DNA strands, which can then be analyzed at any time to get a record of
the cell's activity.
The results of the study were published online on July 27, 2022 in the journal Nature under the title "Recording gene expression order in DNA by CRISPR addition of retron barcodes.
"

Image from Nature, 2022, doi:10.
1038/s41586-022-04994-6
.

For the first step in building Retro-Cascorder, Shipman's team turned to retrons, bacterial sequences that produce specific DNA sequences
when activated.
The authors added a reverse transcript to the gene of interest
.
Each time the gene is activated, the reverse transcript also produces a corresponding DNA sequence
carrying the gene's unique barcode.
Next, these authors want to have a numerator ledger that records these receipts
chronologically.
To do this, they used CRISPR arrays, or long DNA repeats, in which bacteria typically copy the DNA sequences they need in immune memory--- in the order
in which they receive them.

By integrating the CRISPR array and the reverse transcript into the same cell, Shipman's team ensures that every DNA receipt produced by the reverse transcript is inserted into the
CRISPR array.
To retrieve the information contained in the CRISPR array, they simply sequenced the cell's genome and looked at the order
of reverse transcript sequences in the CRISPR array.

8.
Science: Using an improved version of CRISPR to reveal genes that promote emphysema and chronic obstructive pulmonary disease

doi:10.
1126/sciadv.
abo6566

In a new study, researchers from Boston University and Boston Medical Center used improved versions of CRISPR--- CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) to ---understand the function of
genes that promote emphysema and chronic obstructive pulmonary disease (COPD) production.
They found functional outcomes
for these diseases by turning off the expression of genes that promote the pathogenesis of these diseases.
The findings were published in the July 13, 2022 issue of Science Advances, in the paper "CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial.
" cells”
。

An overview of the COPD GWAS gene using the CRISPRi platform from Science Advances, 2022, doi:10.
1126/sciadv.
abo6566
.

Using an improved version of CRISPR, the authors designed a system to turn off the expression of a gene of interest with CRISPRi in iPS cells, or overexpress a gene
of interest with CRISPR activation (CRISPRa).
They grew these cells in a dish and differentiated them into cells
that reside in the lungs.
The type of lung resident cell studied, known as type 2 alveolar epithelial cells (AT2), are progenitor cells in the alveoli--- which are the part of the lungs where gas exchange takes place, and are damaged structures
in emphysema.
So, by understanding how genes found in genome-wide association studies affect AT2 cells, they can begin to understand what these genes might contribute to
diseases that affect these cells, such as emphysema.

Once the AT2 cells were produced, the authors used CRISPRi to turn off the expression of 9 different genes found in genome-wide association studies and analyzed them to understand how these cells were affected, especially their ability to proliferate, which they needed to do when dealing with injuries such as emphysema
.
They noticed that turning off a specific gene, the gene encoding desmoplakin (DSP), caused these cells to proliferate more and increase the expression
of genes involved in cell maturation.
They found that AT2 cells that turned off DSP expression before smoke exposure turned off cell junction gene expression to a greater extent than control cells
.
These cells were also better at forming new colonies than control cells, an indicator
of progenitor cell function.
They then observed mice that had their DSPs removed from lung epithelial cells and compared them
to control mice with normal DSPs.
They found that AT2 cells from DSP-deficient mice were more likely to proliferate after injury, consistent with
findings obtained against human iPS cell-derived AT2 cells.

9.
Nat Biotechnol: Be alert! Clinical therapies based on CRISPR technology may damage the genome of the body's cells!

doi:10.
1038/s41587-022-01377-0

Multiple clinical trials of allogeneic T cell therapy have used site-specific nucleases to interfere with the expression
of T cell receptors (TCRs) and other genes.
Recently, in a study entitled "Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage" published in the international journal Nature Biotechnology, scientists from Tel Aviv University and other institutions said that CRISPR therapy may damage the genome of the body, and CRISPR genome editing methods are very effective.
But it's not always safe, and sometimes the chromosomes that split don't regain their function, and the stability of the genome suffers, which in the long run may lead to cancer
.

In the article, the researchers pointed out the risks associated with the use of CRISPR therapy, which is currently widely used to treat a variety of diseases such as cancer, liver and intestinal diseases, genetic syndromes and other diseases, when investigating the effect of this technique on immune system white blood cells, found that up to 10% of the treated cells will have a significant proportion of the loss of cellular genetic material, this loss may lead to the instability of the cell genome, thereby inducing
。

The researchers said that the first approved CRISPR clinical trial was conducted at the University of Pennsylvania in 2020, when the researchers applied this method in T cells, they obtained T cells from a donor body, then made them express an engineered receptor that targets cancer cells, and used CRISPR technology to destroy the gene encoding the original receptor, which would otherwise cause the T cells to attack the cells in the recipient's body
。 In the current study, the researchers sought to analyze whether the potential benefits of CRISPR therapy might be outweighed by the risks posed by the lysis itself, assuming that broken DNA is not always recoverable
.

Researcher Ben-David explained that the genome in a cell is often broken due to natural causes, but usually it can repair itself, so it generally does not cause damage; However, sometimes specific chromosomes cannot be restored, resulting in the loss of large or even entire chromosomes, and the disruption of this chromosome can destabilize the genome, as we often see in cancer cells, so CRISPR therapy may promote cell carcinogenesis in extreme cases
.

10.
Cell: A new atlas based on CRISPR technology associates each human gene with its function

doi:10.
1016/j.
cell.
2022.
05.
013

The Human Genome Project is an ambitious initiative to sequence
every piece of human DNA.
The project attracted collaborators from research institutions around the world, including the Whitehead Institute, and was finally completed
in 2003.
Now, more than two decades later, Whitehead Institute member Jonathan Weissman and colleagues have gone beyond the human genome sequence in a new study to comprehensively construct the functional map
of genes expressed in human cells for the first time 。 The results of the study were published online in the journal Cell on June 9, 2022, under the title "Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq"
.
They linked each gene to its role in the cell, the result of
years of collaboration on the single-cell sequencing method Perturb-seq.

Image from Cell, 2022, doi:10.
1016/j.
cell.
2022.
05.
013
.

The data can be found on the Weissman lab's website for use by other scientists
.
"It's a big resource, just like the human genome is a big resource, and you can go in and do discovery-based research
," Weissman said.
Instead of determining the biology you're going to study in advance, you can have this genotype-phenotype map that you can go in and sift through without having to do any experiments
.
”

This screening allowed these authors to delve into a variety of biological questions
.
They used it to explore the effects of genes of unknown function on cells, study mitochondrial responses to stress, and screen out genes that cause chromosomal loss or gain, a phenotype
that has proven difficult to study in the past.
Tom Norman, co-corresponding author of the paper and a former postdoc in Weissman's lab, said, "I think this dataset will enable people from other areas of biology to do all kinds of analyses that we haven't even thought of, and suddenly they have data
that they can work with.
" (Biovalley Bioon.
com)