What are the major CRISPR/Cas research or discoveries in the past two months? The editor combed the CRISPR/Cas research news reported in the past two months for everyone to read.
1. Detailed explanation of Science papers! Single-cell lineage tracking based on CRISPR/Cas9 reveals the rate, pathway and driving factors of cancer xenograft metastasis
doi:10.1126/science.abc1944
When cancer is confined to one part of the body, doctors can usually treat it with surgery or other therapies . However, most cancer-related deaths are due to its propensity to metastasize, sending its own seeds (cancer cells), which may take root throughout the body. The exact moment of metastasis is fleeting, mixed in the millions of divisions that have occurred in the tumor. Jonathan Weissman, a member of the Whitehead Institute in the United States, said, "These events are usually impossible to monitor in real time."
Now, in a new study, a research team led by Weissman has turned the CRISPR tool into a tool to achieve this goal. a way. Weissman's laboratory worked with computer scientist Nir Yosef at the University of California, Berkeley and Trever Bivona, a cancer biologist at the University of California, San Francisco, to treat cancer cells in the way that evolutionary biologists view species, drawing extremely detailed family trees. By exploring the branches of this family tree, they can track the lineage of cancer cells to find when a single tumor cell becomes abnormal and spreads its offspring to other parts of the body. The relevant research results were published online in the Science Journal on January 21, 2021. The title of the paper is "Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts".
Picture courtesy of Jeffrey Quinn/Whitehead Institute.
Weissman said, "With this method, you can ask questions like:'How often does this tumor metastasize? Where did the metastasis come from? Where did they go?' By being able to track the history of the tumor in the body, you can reveal The biological difference of tumors , which cannot be observed by conventional means."
2. Interpretation of Cell papers! New research reveals that CRISPR/Cas9 can also be used as a gene editing tool to regulate gene activity.
doi:10.1016/j.cell.2020.12.017
In a series of experiments on bacteria grown in the laboratory, from John Hope, USA Researchers at Kings University have found evidence that the widely used gene-cutting system CRISPR-Cas9 has another function-as a self-regulating switch for the CRISPR-Cas9 gene. It reduces or weakens the effect of CRISPR-Cas9 activity, and may help scientists develop new methods for cell genetic engineering for research purposes. The relevant research results were published online in Cell on January 8, 2021. The title of the paper is "A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression".
Scientists have long been working to unravel the precise steps of CRISPR-Cas9's mechanism of action and how its activity in bacteria can be adjusted up or down. When these researchers were looking for genes that activate or inhibit the CRISPR-Cas9 gene cutting system of Streptococcus pyogenes, they found clues about how this system works.
Specifically, these researchers discovered a gene in the CRISPR-Cas9 system. When inactivated, it will cause the gene editing system to increase its activity in bacteria. The product of this gene seems to be to reprogram Cas9 to act as a brake rather than a "scissors" to lower the activity of the CRISPR system.
3. PNAS: Animal models reveal GPI anchoring defects
doi:10.1073/pnas.2014481118
mental impairment, dyskinesia and developmental delay are typical manifestations of rare diseases caused by GPI protein defects. Researchers at the University of Bonn and the Max Planck Institute for Molecular Genetics used genetic engineering methods to create mice that mimic these patients well. Studies in this animal model show that in GPI-anchored protein defects, genetic mutations can impair the transmission of stimuli in the brain's synapses. These results have now been published in the "PNAS" magazine.
Just like ships anchoring to the seabed in storms and waves, GPI anchoring (GPI = glycosylphosphatidylinositol) ensures that special proteins can remain outside of living cells. If the GPI anchor fails to function normally due to genetic mutations, it will disrupt the signal transmission and transport between cells. Professor Peter Krawitz of the Institute of Genomic Statistics and Bioinformatics at Bonn University Hospital explained: "GPI anchor defects include a group of rare diseases that mainly cause mental retardation and developmental delay."
Mutations in the PIGV gene have been found in most European patients. It encodes an enzyme essential for the synthesis of GPI anchors. Researchers at the Max Planck Institute for Molecular Genetics and their colleagues used CRISPR-Cas9 gene editing technology to modify the PIGV gene in mice based on patient models. Miguel Rodríguezde los Santos of the Charity Institute of Medical Genetics and Human Genetics said: "A large number of behavioral tests have shown that this mouse model very closely reflects the diseases observed in humans."
4.Science sub-Journal: Chinese scientists based on genome-wide screening identified a gene KAT7 promote cell senescence
doi: 10.1126 / scitranslmed.abd2655
In a new study, from the Chinese Academy of Sciences, Chinese Academy of Sciences University, Peking University, and Capital Medical University Xuanwu Researchers at the hospital conducted a CRISPR-Cas9-based genome-wide screen using two types of human mesenchymal precursor cells (hMPC) that exhibit accelerated aging. These two hMPCs are derived from human embryonic stem cells that carry pathogenic mutations in Werner syndrome and Hutchinson-Gilford progeria syndrome, which cause accelerated aging. The relevant research results were published in the journal Science Translational Medicine on January 6, 2021. The title of the paper is "A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence".
These authors identified genes that can reduce cell senescence after deletion, including KAT7. KAT7 encodes a histone acetyltransferase and ranks highest among the two hMPC models of progeria. The inactivation of KAT7 reduces the acetylation of histone H3 lysine, inhibits the transcription of p15INK4b, and alleviates hMPC senescence. In addition, intravenous administration of the lentiviral vector encoding Cas9/sg-Kat7 can reduce the aging of liver cells and liver aging in physiologically aging mice and premature aging Zmpste24-/- mice that exhibit a premature aging phenotype, and prolong lifespan.
5. Nature: Gene editing technology for the treatment of premature aging
doi:10.1038/s41586-020-03086-7
In a recent study, researchers successfully used DNA editing technology to extend the genetic variation associated with premature aging in mice Life expectancy, premature aging is a rare genetic disease that can cause children to age extremely prematurely and may greatly shorten their life expectancy. The research was published in the journal Nature.
Image source: www.pixabay.com.
Progeria, also known as Hutchinson-Gilford Progeria Syndrome, is caused by mutations in the lamina A (LMNA) gene, in which one of the DNA bases C is changed to T. This change will increase the production of the toxic protein progerin, leading to a rapid aging process.
In this study, the researchers used a breakthrough DNA editing technology that replaces a single DNA letter with another DNA letter without damaging the DNA, and further studied how changing this mutation might affect the symptoms of premature aging in mice .
To test the effectiveness of its base editing method, the team initially collaborated with the Progeria Research Foundation to obtain connective tissue cells from patients with progeria. The team used the basic editor of the LMNA gene in the patient's cells in the laboratory setting. This treatment can repair 90% of the mutations in cells.
6. Viruses: Gene-edited mosquitoes can help prevent the spread of
Zika virus doi:10.3390/v12111231
At present, a method to prevent the spread of Zika virus has been approved by the U.S. Environmental Protection Agency (EPA). This method will be released in 2021. More than 750 million genetically modified mosquitoes will be released to Florida Keys in 2022. These "suicidal mosquitoes" have undergone genetic changes and cannot produce offspring, or their offspring cannot survive to adulthood, and therefore lose the ability to spread diseases. However, the removal of offspring of mosquitoes may complicate the environment, such as disrupting the food chain. A new study from the University of Missouri offers another option: genetically modify mosquitoes to make them completely resistant to Zika virus.
Alexander Franz, associate professor of the School of Veterinary Medicine at the University of Missouri, used CRISPR gene editing technology to collaborate with researchers at Colorado State University to produce mosquitoes whose Zika virus cannot replicate in their bodies, so they cannot infect humans by biting.
Franz said: "By inserting artificial genes into their genomes, we trigger an immune pathway to recognize and destroy the Zika virus RNA genome. By developing these virus-resistant mosquitoes, the chain of disease transmission is blocked. Therefore, it is no longer possible to spread to humans.”
Franz added that this genetic modification is heritable, so offspring mosquitoes will also become resistant to Zika virus.
7. AJHG: Research reveals the gene mutation behind Lynch syndrome
doi:10.1016/j.ajhg.2020.12.003
Colorectal cancer is the third most common form of cancer. Although 90% of cases are in people over 50 years old, there is still a high incidence among young people, and the reasons are still unexplainable. Family history is one of the high-risk factors for the development of colorectal cancer, and it is usually recommended that people with such a history have more frequent screening tests or start screening than the recommended 45-year-old age. People with a family history of cancer usually use genetic testing to look for mutations associated with cancer risk. However, these tests do not always provide useful information.
In a new paper in the American Journal of Human Genetics, Dr. Jacob Kitzman of the Department of Human Genetics of the Michigan Department of Medicine and a group of authors describe a method of screening for so-called genetic variants that are found in humans. Hope to find out mutations that may cause disease. To this end, they referred to a genetic disease called Lynch syndrome, also known as hereditary non-polyposis colorectal cancer. Like BRCA1, some genes behind Lynch syndrome are well described. However, "the genetic variation that may exist in the gene associated with Lynch syndrome is basically unknown to scientists," Kitzman said.
The research team used a technique called deep mutation scanning to measure the impact of mutations in the gene MSH2, which is one of the main causes of Lynch syndrome. They used CRISPR-Cas technology to delete the normal copy of MSH2 from human cells and replaced it with a library of every possible mutation in the MSH2 gene. This creates a mixture of cells, where each cell carries a unique MSH2 mutation. This cell population is treated with a drug called 6-thioguanine, and this chemotherapy kills only cells with functional variants of MSH2.
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February 2021:
1. Science: CAR-T cells edited by CRISPR genes are safe in cancer patients
doi:10.1126/science.aba7365; doi:10.1126/science.aba9844
In a new study, from the University of Pennsylvania and Stanford University The researchers of, combined two state-of-the-art methods---CRISPR (editing DNA) and T-cell therapy (using the sentinels of the immune system to destroy tumors)---to pioneer the rapidly evolving field of cancer immunotherapy A new chapter has been opened. They reported that two women and one man, both over 60 years old, had a sarcoma and the remaining two had a type of blood cancer called multiple myeloma. The three patients received their own CRISPR gene-edited immune cell therapy last year. The relevant research results were published online in the journal Science on February 6, 2019. The title of the paper is "CRISPR-engineered T cells in patients with refractory cancer".
Picture from Science, 2020, doi:10.1126/science.aba9844.
For these three patients, the benefit is limited: one has died, and the other two have worsened. However, Carl June, the corresponding author of the paper and a cancer researcher at the University of Pennsylvania, said that this clinical trial, which has undergone years of regulatory review, is not intended to cure cancer. On the contrary, its goal is to show that this strategy is feasible and safe.
The researchers first looked for patients whose tumors produced a protein called NY-ESO-1, in order to add a gene encoding this protein to T cells extracted from these patients. These patients also need to carry a specific type of human leukocyte antigen (HLA), which is an immune protein complex that helps the T cells perfused back into the patient's body to thrive. The four eligible patients are all very sick, which is often encountered with this new therapy. A female patient with multiple myeloma received three bone marrow transplants. Another female patient who suffered from sarcoma in her thirties was too sick to receive her genetically modified T cell therapy in the laboratory. She received clinical care and died.
In order to promote T cells from these patients to resist the disease they are suffering from, these researchers used CRISPR to knock out two genes encoding the so-called T cell receptor (TCR). In addition, they also weakened the third gene, which encodes a protein called PD-1. They speculated that PD-1 can block the immune response, and the effects of clearing PD-1 may enrich the function of T cells. Subsequently, they inserted a different gene encoding a T cell receptor targeting NY-ESO-1 into T cells.
Intensive monitoring of these three patients, including blood draws to study the genetically modified T cells in their bodies, confirmed that CRISPR causes some off-target changes. But they are very few, and the number of cells with these unexpected DNA changes will gradually disappear over time. Encouragingly, these CRISPR gene-edited T cells can last for at least 9 months in the body, while in existing CAR-T cell therapy studies, this number is about 2 months. Imaging examinations showed "good healthy T cells". In laboratory studies, they can fight off cancer within a few months after being infused back into the patient's body.
But in these three patients, the prognosis was not high. The best response was observed in a sarcoma patient whose primary tumor shrank, but his cancer later deteriorated. These researchers put forward possible reasons, including the small number of patients receiving treatment, the possible limitations of using NY-ESO-1 as a target (the choice of it as a target is partly due to its good safety record) and failure Can knock out all three genes in many T cells.
2. Sci Rep: Carrying Parkinson's disease mutant cells is helpful for disease research
doi:10.1038/s41598-020-60273-2
In a recent study, scientists used gene editing tools to introduce disease-related gene mutations into monkey-derived stem cells In, and successfully inhibited Parkinson’s disease patients often have abnormal cell biochemical reactions. The author of the article, Professor Marina Emborg of the University of Wisconsin-Madison, said: "We now know how to insert a single mutation (point mutation) into monkey stem cells." The results were published in the recent "Scientific Reports" magazine. In this study, these researchers used the gene editing technology CRISPR to change a single nucleotide in the cell's genetic code and named it G2019S.
In humans with Parkinson's disease, this mutation causes the enzyme LRRK2, which is involved in cell metabolism, to be overactive. For the first time, this new study has produced cells that only have the G2019S mutation, which makes it easier to study the role of the mutation in disease.
3. Science Sub-Journal: The gene editing tool CRISPR-Cas9 has encountered new setbacks! New research reveals that it can cause a lot of unwanted DNA duplication
doi:10.1126/sciadv.aax2941
In a new study, researchers from the University of Münster in Germany found that in mice undergoing routine CRISPR-Cas9 gene insertion, there is a high frequency of unnecessary DNA repetitions. The relevant research results were published in Science Advances on February 21, 2020. The title of the paper is "Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events". They described how they found unnecessary DNA duplications and reminded other researchers about this.
This discovery was accidental, because as part of the immunological research work, they were studying a calcium binding protein encoded by the gene S100A8. To this end, they used CRISPR-Cas9 to make the gene unable to express the protein, which is a form of knockout editing (that is, the use of CRISPR-Cas9 gene editing to remove target genes). They first perform standard PCR tests and then perform more professional PCR tests to detect target genes to ensure that everything goes according to plan. The results of the study showed that only two edits were successful, which surprised them. They then bred a successfully gene-edited mouse with a wild mouse to understand why the editing success rate was so low. Testing of mouse offspring using a special type of PCR revealed that seven offspring of mice carry the edited gene S100A8, and the remaining offspring of mice have unwanted DNA duplications.
The researchers were shocked by their findings, so they conducted a second study, editing a different gene in mice. Professional tests showed that among the 50 mice tested, 30 mice had multiple copies of unwanted genome fragments, and these copies were inserted into the mouse genome as part of CRISPR-Cas9 editing. The experiment once again showed that the standard PCR test failed to find these copies.
4. Cell Stem Cell: The use of base editors can cure genetic diseases in human cells
doi:10.1016/j.stem.2020.01.019
The genome editing tool CRISPR/Cas9 developed in 2012 can cut off the mutated fragment in the gene and replace it with a non-mutated fragment, which is called a base editor The new CRISPR can repair mutations without cutting DNA. Therefore, the use of base editors for genome editing is considered safer. Now, in a new study, researchers from research institutions such as the Utrecht Research Institute and Utrecht University in the Netherlands have confirmed for the first time that base editors can safely cure cystic fibers in patient-derived stem cells化 (cystic fibrosis). The relevant research results were published online on February 20, 2020 in the journal Cell Stem Cell. The title of the paper is "CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank". The corresponding authors of the paper are Hans Clevers of the Utrecht Institute and Jeffrey Beekman of Utrecht University.
Picture from Cell Stem Cell, 2020, doi:10.1016/j.stem.2020.01.019.
According to Maarten Geurts, a biologist at the Utrecht Institute and Eyleen de Poel, a biologist at Utrecht University, a new CRISPR enzyme developed in 2018 makes CRISPR technology more precise and less error-prone. Maarten said, “In the traditional CRISPR/Cas9 genome, cutting a specific piece of DNA can cause DNA damage. The purpose of this is that the cell uses the'healthy' DNA segment made in the laboratory to repair this cut. In the new CRISPR technology of the base editor, Cas9 has been improved so that it no longer cuts the DNA, but can still detect the mutation site. Therefore, there is no need to cut the DNA and replace the defective DNA fragment. The mutation site can be Direct on-site repair makes it a more effective genome editing tool." The
current new research shows that this new version of CRISPR/Cas9 (the base editor) can be safely and effectively applied to human stem cells.
5. APL Bioeng: Use CRISPR to open DNA to eliminate diseases
doi: 10.1063/1.5127302
A protein editing cofactor is clearing the way for cut and paste DNA editors (such as CRISPR) to access previously inaccessible genes of interest. Opening up these areas of the genetic code is essential to improve the efficiency of CRISPR and move toward future, gene-based disease treatments. This DNA binding editing cofactor was designed by an American who described their design in APL Bioengineering.
Lead author Karmella Haynes from Arizona State University and Emory University said: "The innovation of this paper is that it uses another protein that is delivered in conjunction with the CRISPR DNA editor and removes the chromatin packaging so that CRISPR can Get DNA more easily."
They used a sophisticated artificial system that can open or close the chromatin packaging of a gene-the luciferase gene-which encodes an easily detectable luminescent protein. When examining the filling state of chromatin, the research team discovered several editing assistants, called DNA-binding transiently expressed activation-associated proteins (AAPs), which destroyed chromatin and enabled CRISPR to successfully edit the luciferase gene.
6. Gene editing Daniel Zhang Feng uses the CRISPR-Cas13-based SHERLOCK system to detect the coronavirus 2019-nCoV
News source: A protocol for detection of COVID-19 using CRISPR diagnostics The
most recent novel coronavirus SARS-CoV-2 (previously known as 2019- nCoV, the disease caused by this virus infection is called COVID-19) The epidemic has brought huge challenges to global health. In response to this global challenge, the Broad Institute, McGovern Institute for Brain Science and their partner institutions are committed to providing potentially useful information, including sharing information that may support the development of potential diagnostic methods.
As part of the response measures taken, Feng Zhang, Omar Abudayyeh, and Jonathan Gootenberg developed a research protocol suitable for purified RNA, which may help to develop a CRISPR-based diagnostic method to clinically detect 2019-nCoV. This research plan includes three steps. It can be used to test RNA currently extracted from clinical samples for quantitative PCR (qPCR) testing: Step 1: Amplify the extracted RNA isothermally at 42°C for 25 minutes; Step 2: Let the Incubate the amplified product with Cas13 protein, gRNA and reporter molecule at 37°C for 30 minutes; Step 3: Immerse the test strip in the reaction product solution in Step 2, and the result should appear within 5 minutes.
This initial research protocol is not a diagnostic test method and has not yet been tested on patient samples. Any diagnostic method needs to be developed and validated for clinical use, and it needs to follow all local regulations and best practices. Nevertheless, this research program still provides a basic framework for using test strips to establish a SHERLOCK-based 2019-nCoV diagnostic method.
7. Nature: 351 new giant phages were discovered from different environments on the earth, they blur the line between virus and bacteria
doi:10.1038/s41586-020-2007-4
In a new study, from the University of California Berkeley, Colorado State University, Stanford University, U.S. Department of Energy Joint Genome Research Institute, University of Pittsburgh School of Medicine, Sun Yat-sen University, China, University of Cape Town, South Africa, French National Research Center, University College London, University of Melbourne, Australia, Danish Technology Researchers from universities, the Japanese Atomic Energy Agency, and the University of Toronto in Canada have discovered hundreds of unusually large viruses that can kill bacteria. They usually have functions related to living organisms, which blur the relationship between living bacteria and viruses. Boundaries. The relevant research results were published online on February 12, 2020 in the journal Nature, and the title of the paper is "Clades of huge phages from across Earth's ecosystems". The corresponding author of the paper is Professor Jill Banfield of the University of California, Berkeley. The first authors of the paper are Basem Al-Shayeb, a graduate student at the University of California, Berkeley, and Rohan Sachdeva, a research assistant.
These researchers discovered these huge phage (huge phage, also known as megaphage) by searching huge DNA databases. These DNA databases range from nearly 30 different global environments --- from the intestines of premature babies and pregnant women to Tibetan hot springs. , Wards, oceans, lakes and deep underground---produced. In total, they identified 351 different giant phages whose genomes were four or more times larger than the average genomes of viruses that swallow single-celled bacteria. Among them, there is the largest bacteriophage discovered so far: its genome is 735,000 bases (735kb), which is nearly 15 times larger than the average bacteriophage genome. The largest known phage genome is much larger than the genomes of many bacteria.
Ironically, in the DNA carried by these giant bacteriophages, there is a part of the CRISPR system that bacteria use to fight viruses. It is very likely that once these phages inject their DNA into the bacteria, this viral CRISPR system will enhance the CRISPR system of the host bacteria, and it is likely that the bacterial CRISPR system will target other viruses.
One of these giant phages can also make a protein similar to the Cas9 protein. Cas9 was developed by Jennifer Doudna of the University of California, Berkeley and her European colleague Emmanuelle Charpentier, a revolutionary tool for gene editing, CRISPR-Cas9. Part. These researchers call this tiny protein CasØ, because the Greek letter Ø or phi is often used to mean bacteriophages.
Sachdeva said, “In these huge phages, there is great potential for finding new tools for genome engineering. Many of the genes we discovered are unknown, they have no hypothetical function, and may be new proteins in industrial, medical or agricultural applications. Sources."
8. Sci Adv: CRISPR gene editing can repair hereditary liver damage
doi:10.1126/sciadv.aax5701
Recently, researchers from the University of Pennsylvania School of Medicine published online studies in the journal Science Advance that a new CRISPR gene editing technology can prevent the occurrence of a hereditary liver disease driven by hundreds of different mutations, and Improved the clinical symptoms of mice. The results of the study show that this promising CRISPR tool can potentially treat patients with rare metabolic abnormalities in the urea cycle caused by ornithine transcarbamylase (OTC) deficiency and other mutations at different sites of the same gene.
This CRISPR gene editing method is based on the method developed by the same research team before. This time, the researchers used a new type of dual adeno-associated virus (AAV) to deliver its active ingredients, by inserting "minigenes" into the genome to achieve sustained expression of OTC in liver cells. Compared with previous treatments to correct single-point mutations, this "cut"-"paste" method can significantly improve the clinical performance of newborn mice and can continue into adulthood.
"Like most genetic diseases that have fatal effects on newborns, long-term effective early treatment is essential." said the author of the article, Dr. James Wilson, Director of Gene Therapy Program and Gene Therapy. “Here, we have further improved the CRISPR technology to not only maintain the expression of OTC in cells, but also expand its therapeutic capabilities. Our goal is to finally transform this gene editing method to the clinical stage to treat patients with OTC disorders and Patients with other genetic diseases have mutations scattered throughout the gene, rather than a single mutation."
9. The latest two Chinese scientists Nature Biotechnology have constructed an ultra-precise base editor
doi:10.1038/s41587-020-0414-6; doi :10.1038/s41587-020-0412-8
CRISPR-based gene editing has potential therapeutic advantages, but there are also some technical defects. Among these gene editing tools, the base editor can rewrite the four bases that make up DNA---adenine (A), cytosine (C), thymine (T) and guanine (G)--- one.
Now, in two new studies, researchers from the Broad Institute and the Howard Hughes Medical Institute have invented new CRISPR tools that improve the accuracy of base editors and genome targeting capabilities. Solved some of the challenges they faced. The relevant research results were published online on February 10, 2020 in a paper in the journal Nature Biotechnology, the paper titles are "Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors" and "Continuous evolution of SpCas9 variants" compatible with non-G PAMs"
Picture from Frontiers in Plant Science, 2018, doi:10.3389/fpls.2018.01361.
In the first new study, these researchers designed a new cytosine base editor that reduced an elusive off-target editing by 10 to 100 times, making these new cytosine base editors special It is expected to be used to treat human diseases. In the second new study, they obtained a new generation of CRISPR-Cas9 protein by evolving the existing Cas9 protein, which can target a larger number of pathogenic mutations, including a mutation that causes sickle cell anemia. . Prior to this, this mutation was still difficult to target with previous CRISPR methods.
The corresponding author of these two papers, the director of the Merckin Institute for Medical Transformation Technology, the Broad researcher, the professor of chemistry and chemical biology at Harvard University, and the researcher of the Howard Hughes Medical Institute David Liu said, “Given that the era of human genome editing is still in It is a fragile initial stage, so when we start to introduce these gene editors into the human body, it is important that we do everything we can to minimize the risk of any adverse effects. This elusive off-target editing--- Cas9-independent edit (Cas9-independent edit, that is, does not rely on Cas9 editing)-to minimize is an important step to achieve this goal. This off-target editing can occur at random locations in the genome. When you do In 10 experiments, you will get 10 different answers. This makes researching this extremely challenging."