Mathematical models of gene editing using CRISPR

Mathematical models of gene editing using CRISPR

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One confusing thing I have found when reading articles about possible CRISPR based gene therapy treatments in humans is that there is vey little discussion about what percentage of your cells will actually have their DNA edited, the rate at which the editing takes place, and how to quantitatively estimate how many edited cells would be needed to "cure" a specific disease. (It's not so confusing how edits could be made to a small embryo, what's confusing is what is actually possible for grown patients)

Can someone provide a few basic facts that calibrate what is currently possible and what is needed.

For instance, is it currently possible to edit more than 50% of the cells of an adult nematode (or more complicated organism) using CRISPR? How long does it take (minutes, hours, days, weeks).

For the current list of human diseases that are possible targets for gene therapy, how many cells need to be edited for one of those diseases to be "cured" (hundreds, thousands, billions, etc)

But in general is there a name for the mathematical models that try to predict how many cells will end up being edited, how those edited cells will multiply and replace diseased tissue, and how many edits are needed to generate a concentration of missing protein for a treatment to be successful?


There are several probabilities that you need to take into account.

  • The efficiency of DNA transformation of CRISPR encoding DNA and target DNA into the host cell (varies between cell types and transformation agent).
  • Efficiency of cutting by CRISPR at target site. (varies by DNA compaction - which varies by site and cell type, and guide RNA)
  • Number and cutting efficiency at off targeting sites.
  • Efficiency of degradation of free DNA by cellular antiviral systems (varies between cell types)
  • Efficiency of DNA repair machinery to fix dsDNA breaks. (thus removing CRISPR cut sites. Varies by cell type)
  • Efficiency of recombination machinery (homologous recombination and microhomologous recombination) (which is used to insert new DNA sequence).
  • Probability of NHEJ damaging target site. (which can delete the CRISPR cut site)

Too many things to count.

However to give you some idea. The efficiency of CRISPR making a correctly targeted knock in for DT40 (chicken lymphoma) is ~1% of all resistant colonies (ie colonies that have taken up the DNA seq used to make the knock in), when lipofectamine reagents are use. Lipofectamin trasfromation efficiency varies depending on the size of the transformed DNA, host cell type and plating density. So for a 5kb construct that transformation efficiency varies between 30%-50%.

CRISPR is nearly 100% efficient in yeast cells, ie all knock in cells are correctly targeted. This is likely due to the high levels of HR in yeast



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In the summer of 2013, John Schimenti, Biomedical Sciences and Molecular Biology and Genetics, began trying out a new, up-and-coming genetic-editing method known as CRISPR. In just a couple of months, he saw how easily the technique worked on mouse models. “The power was self evident, and it was so incredibly straightforward,” he says.

How CRISPR Works

CRISPR, which stands for “clustered regularly interspaced short palindromic repeats,” is a DNA sequence found in bacteria. The discovery of CRISPR dates back to the 1980s, when researchers at Osaka University sequenced the genomes of common bacteria. They found the repeating DNA sequences in many species but did not know its biological purpose.

Years later, scientists in the dairy industry confirmed that bacteria use CRISPR to destroy viruses. The molecular system works by incorporating “spacer” DNA sequences that match the DNA of viruses that have previously attacked the bacteria and its ancestors. When a virus attacks, RNA made from the CRISPR DNA binds to the matching viral DNA by Watson-Crick base-pairing. The RNA also binds to a DNA-cutting protein called a nuclease, and the formation of the whole complex results in the viral DNA being chopped up and destroyed. If an unknown virus attacks, the CRISPR system makes new spacer sequences to protect it against the virus in the future. With this knowledge in hand, the dairy industry identified different strains of bacteria and could see whether bacterial cultures used in products such as yogurt or cheese were immune to specific viruses.

It wasn’t until 2012, however, that researchers discovered a way to leverage the CRISPR system to slice up any DNA sequence in bacteria, viral or not. The type of CRISPR system they used involved a nuclease called Cas9. The researchers could engineer the CRISPR DNA sequence to make an RNA that matched essentially any DNA target they wanted. The RNA bound to Cas9 would do the rest, finding the matching DNA sequence and cutting it.

Since then, researchers across the globe have worked at an incredible pace to use CRISPR for further applications. Scientists have demonstrated that the CRISPR-Cas9 system works in a wide range of organisms and cells, including human cells, plants, and model organisms such as flies, worms, and mice. Many Cornell scientists, in particular, are embracing the cutting-edge technology and testing its limits.

Reproduction Genetics

Schimenti was among the first at Cornell to utilize CRISPR in his research. Today, he uses the technique to better understand reproduction genetics. His lab is currently looking at sequenced human DNA and identifying genes that are bioinformatically predicted to harm reproduction, specifically those that affect meiosis. Schimenti and his lab are able to use the CRISPR technique to target equivalent genes in mice as a model to understand human fertility.

CRISPR is the newest of several tools available for precision genome editing, others include zinc finger nucleases, homing endonucleases, and more recently, TAL effector nucleases (TALENs). What makes CRISPR so transformative is its simplicity and how quickly it can generate a CRISPR for a new target. “What we do with CRISPR could be done already using embryonic stem cells, but that was much more difficult,” says Schimenti. “With CRISPR, it takes no more than two to three days from designing your mutation, to having your reagent, to making the mouse. This is just unbelievable. We can make very, very precise mutations with ease and even though my lab only does this with mice, the other huge thing that this technology does is work in virtually all life forms.”

Creating Fruit Fly Models

CRISPR has especially had an impact on research using the model organism drosophila, more commonly known as the fruit fly. Prior to gene editing, researchers using drosophila made mutant flies—flies with a specific genetic change relevant to a study—somewhat at random. “You had to come up with a genetic screen or method to find that one mutation in that one fly in a large population of flies with many other mutations,” says Daniel Barbash, Molecular Biology and Genetics. “Technologies like CRISPR allow us to go in and design the mutation we want and know where it is.”

The technology’s levels of power, simplicity, and precision have completely transformed the way researchers can study genetics in fruit fly populations. Barbash’s lab works on interspecific hybrids of drosophila, for example, a cross between D. melanogaster (the “lab rat” of flies) and its sibling species D. simulans. The researchers are looking at genetic mutations to determine whether a hybrid species of fly will live. The goal is to understand how these genes work in each species.

CRISPR not only makes it possible to create genetic mutations in various species of drosophila for which genetic editing was previously difficult, it also speeds genetic editing up to the point where researchers can perform studies they hadn’t considered before.

“Before CRISPR, no one would have thought of doing an experiment looking at the effect of a single mutation in 50 to 100 different populations of drosophila because there was just no practical way to do it,” Barbash explains. “Once you see this technique, a light bulb goes off and you can do that. It will give us a much richer view of how individual genes interact with the rest of the genome in real, natural populations.”

Given the newness and ongoing development of the technology, researchers are still learning how to apply CRISPR in their labs. Barbash says that the drosophila community at Cornell has met numerous times to collaboratively implement CRISPR techniques across campus. The group shares information about different approaches, and labs are helping one another learn the best specific protocols for various use cases.

Chun Han, Molecular Biology and Genetics, is in the process of establishing CRISPR protocol in his lab. Han, too, uses drosophila and says that it has been useful to talk to other researchers on campus, such as Barbash, who are already using the tool.

Han studies dendrite morphogenesis, the process in which nerve cells establish a complex network of branches, and dendrite degeneration, the process in which these nerve cell branches degrade. Both are important in preventing inflammation and keeping tissue stable, in both flies and humans.

Unlike more traditional uses of CRISPR, in which researchers use the tool to slice up a specific DNA sequence, Han is interested in using CRISPR to modify fly genes that could play a role in neurodevelopment. For example, he would like to place a fluorescent tag on a gene to trace it within a cell. This tagging is possible by overexpressing a type of protein in the tissue, but this method can cause unwanted phenomenon, Han explains. “By using CRISPR, we modify the gene in a way that maintains the natural physiological levels,” he says. “It’s much more relevant to a normal situation.”

CRISPR versus TALENs in Plant Sciences

Adam Bogdanove, School of Integrative Plant Science, is one of the leading scientists in TALENs, a gene-editing tool that, prior to the advent of CRISPR, prompted Nature Methods to cite gene editing as “Method of the Year” in 2011. Much like CRISPR, TALENs make it possible to target any specific DNA sequence in virtually any organism. Each tool, however, has its different advantages.

“CRISPR-Cas9 has a lower barrier to entry, and it’s quick,” says Bogdanove. “We, in fact, use CRISPR in some cases to study TAL effectors and their targets in plants.” TAL effectors are the proteins that are engineered to make TALENs. In nature, TAL effectors are used by plant pathogenic bacteria to change host gene expression and cause disease.

“Before CRISPR, no one would have thought of doing an experiment looking at the effect of a single mutation in 50 to 100 different populations of drosophila, because there was just no practical way to do it,” Barbash explains. “Once you see this technique, a light bulb goes off.”

That said, Bogdanove points out that CRISPR has some disadvantages compared to TALENs. CRISPR, in its current state, has more off-target effects, meaning that it can sometimes cut genes that it is not designed to affect. In a research setting, this isn’t a big problem, since a researcher can perform control experiments to identify and account for any off-target effects. But when it comes to applications such as gene therapy and agricultural biotech, there is no room to allow for off-target effects. Under these circumstances, TALENs might be preferred.

Bogdanove says that CRISPR and TALENs are both “revolutionary technologies that Cornell is pushing the frontiers of, especially in plant sciences.” The School of Integrative Plant Science, led by Alan Collmer, is embracing these technologies to push plant biology and translational research that will bring biology into agricultural fields.

“It seems that the limiting factor going forward will not be the technology it will be how we use it,” says Collmer. “There are a lot of people at Cornell who have deep knowledge about what kind of changes in plants would really help people, whether it’s nutrition or soil health or pathogen and insect resistance. These are all huge problems that are very real and are now addressable with plants with CRISPR and TALENs.”

Unlike previous methods of genetic editing, CRISPR and TALENs don’t leave behind foreign DNA. “It’s totally natural in the sense that we could limit ourselves, for public peace of mind, to what is naturally available within the variation of a given crop and its relatives,” says Collmer. Unlike traditional plant breeding, using these genetic-editing tools speeds up the link between discovery and translational benefit. “There is a huge effort in the plant sciences now to leverage genomics to associate genes with phenotypes,” says Collmer, which means that Cornell is in a good position to translate this knowledge into better plants and crops.

One researcher who is exploring the agricultural applications of CRISPR is Kenong Xu, School of Integrative Plant Science, Horticulture. Xu works on discovering and characterizing apple genes of horticultural and economic importance. For example, he is interested in seeing whether CRISPR can be used to modify an apple’s acidity gene. He says that, to date, he has not heard of any other scientists applying CRISPR to apples, and he thinks that the acidity gene is a good target to test whether the technology can work in the fruit.

The acidity gene is especially important to apples because, in most cases, each apple variety carries two copies of the gene. One copy is functional and the other is nonfunctional because of a mutation in the gene. When making crosses between apples, the expectation is that one-fourth of the apples will carry two malfunctioning genes. These apples have very low acidity and are therefore unacceptable in taste and have no commercial value. “You really need to remove them, and you don’t need to plant them in the orchard. Apple seedlings take several years to bear fruit, so if you have hundreds of progeny, one-quarter is a significant portion to lose,” Xu says.

“The advantage of CRISPR and TALENs compared to traditional biotechnology is that they are much more precise,” says Xu. “It’s very desirable to have a tool to manipulate important genes, including the fruit acidity gene, in their native genomic environment for better fruit.”

CRISPR at the Molecular Level

Ailong Ke, Molecular Biology and Genetics, works to better understand the CRISPR system at the molecular level. Ke specifically studies the type I CRISPR system. The CRISPR-Cas9 system currently used as a gene-editing tool is the type II CRISPR system. It’s simple and powerful because the Cas9 protein performs both the targeting and the degradation activity.

Type I CRISPR, instead, involves a large complex of proteins, called the cascade, and the Cas3 protein. The cascade targets the DNA it wants to cut, and the Cas3 protein performs the degradation activity.

“I would argue that there could be more exciting applications from the type I system,” says Ke. “The Cas9 system’s base complementarity is roughly 20 nucleotides. In the type I system it’s 30 to 35, so it’s targeting a longer stretch of DNA. Targeting and degradation happens in two steps, so it is perhaps a more regulated process and could be more specific and better controlled.”

For now, however, Ke is focused on better understanding how the type I CRISPR system works. So far, he and his lab have obtained a structure of the cascade using electron microscopy and a crystal structure of the Cas3 protein. The work on the Cas3 protein was published in Nature Structural & Molecular Biology in September 2014. Both structures capture a snapshot of how the system is working. The goal is to capture multiple snapshots to create a sort of “molecular movie” of activity.

Ke explains that the next steps are to capture more crystal structures of Cas3 and to perform single molecule experiments, meaning experiments on specific molecules that track their activity through a particular event. For example, it could demonstrate how the cascade recognizes DNA in incremental steps, how the cascade recruits Cas3, and then, how it moves away. With this information, Ke says they will be able to understand the details of the system.

A Durable Technique

What’s clear is that the CRISPR system is here to stay, and Cornell scientists are embracing the latest technology. “It’s a really powerful technology and there’s got to be more powerful applications coming out of that,” says Ke. “It will become more efficient and different from what it is now. It just takes imagination.”

“It’s constantly being tweaked. We’re talking about a technology that’s only been around for a year and half,” Schimenti says. “There are literally thousands of scientists who have jumped on this—it’s being improved all the time—and people at Cornell are part of this community.”

Schimenti is also the Director of Cornell’s Stem Cell and Transgenic Core Facility, supported by NYSTEM. The facility recently added CRISPR services, for use in mouse models only, for the wider Cornell community, speeding along the adoption of the technology. CRISPR has quickly become the most requested service, Schimenti says. He adds that requests come from researchers across a variety of projects, from basic science to clinical research done at Cornell Weill.

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1. Introduction

For many years, molecular biologists have sought ways to use cellular repair mechanisms to manipulate DNA through genome editing. In this way, they would have the power to change the genome by correcting a mutation or introducing a new function (Rodriguez, 2016). For this purpose, genome editing technologies were developed (Memi et al., 2018). In recent years, clustered regularly interspaced short palindromic repeats technology (CRISPR-Cas9) has become the most preferred method of gene editing. This technology has advantages such as high accuracy, easy handling, and relatively low cost compared to previous technologies, such as zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Thanks to these benefits, CRISPR-Cas9 technology can be easily applied in any molecular biology laboratory.

Genome editing technologies are used in the formation of human disease models in experimental animals and for the understanding of basic gene functions. They also have great therapeutic potential for future treatment of untreated diseases such as certain cancers, genetic disorders, and HIV/AIDS. Today, genome editing in somatic cells is one of the promising areas of therapeutic development (Otieno, 2015). However, various bioethical issues have arisen due to the potential impact of these technologies on the safety of food stocks and clinical applications (Hundleby and Harwood, 2018 Hirch et al., 2019). This review discusses the challenges, possible consequences, and bioethical issues of CRISPR-Cas9 in detail.

Lesson Plan: Gene Editing using CRISPR

As a high school or undergraduate Biological Sciences teacher, you can use this set of computer-based tools to teach about CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats, a new gene editing technology that could enable certain species to adapt to the impacts of climate change.

This lesson plan includes resources that teach about gene editing using the CRISPR-Cas 9 pathway in bacteria. This pathway is a part of the adaptive immunity against phage infection in bacteria. It can be engineered to be used as a gene editing tool in living organisms. This lesson plan includes case studies that show how CRISPR gene editing technology can be used as a climate adaptation strategy.

Thus, the use of this lesson plan allows you to integrate the teaching of a climate science topic with a core topic in Biological Sciences.

Teacher-contributed lesson plan by Dr Sneha Bhogale, Pune, India.

Want to know more about how to contribute? Contact us.

Use this lesson plan to help your students find answers to:

  1. What is the function of CRISPR in bacteria? Describe the main components of the CRISPR-Cas9 system.
  2. Describe the two main DNA repair mechanisms in a cell.
  3. Explain how the CRISPR gene editing technique exploits the cell’s DNA repair system to introduce targeted mutations.
  4. How can CRISPR gene editing help plant breeding programs to adapt to the effects of climate change? Elaborate using a suitable example.
  5. Discuss the use of CRISPR technology as a climate adaptation strategy to conserve coral reefs.

About Lesson Plan

DNA Repair Mechanisms, Double Stranded Breaks (DSBs)

Non- Homologous End Joining (NHEJ)

Homologous Recombination (HR)

Targeted Mutations, Nucleases

Climate Mitigation and Adaptation

Here is a step-by-step guide to using this lesson plan in the classroom/laboratory. We have suggested these steps as a possible plan of action. You may customize the lesson plan according to your preferences and requirements.

Step 1: Topic introduction and discussion

  1. Begin with introducing what gene editing is and explain how it is different from genetic engineering- Gene editing, is a process in which DNAis inserted, deleted, modified or replaced at a specific site in the genome of a living organism. Genetic engineering, on the other hand randomly inserts or deletes genetic material to introduce mutations.
  2. In gene editing, nucleases/ molecular scissors are used which introduce a double stranded break (DSB) in the DNA at specific locations after which DNA repair mechanisms of the cell take over resulting in targeted mutations (edits).
  3. Then, briefly discuss the commonly used nucleases- meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENS) and CRISPR- that are used for gene editing.
  4. Emphasize that this lesson plan will focus on the CRISPR-Cas9 system of gene editing, as it is reported in recent times to be more efficient and effective than the others.
  5. Use this animated video, ‘Genome Editing with CRISPR-Cas9’, narrated by Feng Zhang, McGovern Institute of Brain Research, MIT, to introduce the topic of gene editing using CRISPR-Cas9 system and to briefly describe the structural components of the CRISPR-Cas9 pathway.

Step 2: Extend understanding of the CRISPR-Cas9 pathway and CRISPR gene editing using an interactive visualization

  1. Use the interactive visualization, ‘CRISPR-Cas9 Mechanism & Application’ by Howard Hughes Medical Institute (HHMI) BioInteractive, to enable your students to visualize how the CRISPR-Cas9 technology works at the molecular level and to explore its different components.
  2. Start by launching the ‘interactive’ component of the visualization tool.
  3. Navigate through the visualization to sequentially describe the gene-editing events of targeting and binding of the CRISPR-Cas9 complex to the target DNA, cleaving or breaking of the DNA at the target location and repairing of the DNA to introduce the desired mutation.
  4. Use the ‘explore’ button at every step to describe the different molecular components involved in the pathway.
  5. Use the tab, ‘How it’s used’ to view 20 short videos that explain how CRISPR gene editing technology can be used to achieve different results in its applications in science and industry.

Step 3: Discuss two case studies where CRISPR gene editing has been used as a climate adaptation strategy

  1. Use the video, ‘Gene editing yields tomatoes that flower and ripen weeks earlier’ by Zachary Lippman, Cold Spring Harbor Laboratory (CSHL), to describe his use of CRISPR gene editing in two varieties of tomato plants to make them flower and ripen earlier than usual.
  2. Use the video to explain how this approach is useful to obtain faster and higher yields of the tomato crop.
  3. Discuss, using the video how this will also enable plants to be grown in higher latitudes, thereby offsetting crop loss, if any, due to global warming.
  4. To enable better understanding of Dr Lippman’s work, direct your students to listen to a CSHL Base Pairs podcast, link to which is available in the additional resources section of this lesson plan.
  5. Use the reading, ‘CRISPR used to genetically edit coral’ by Hanae Armitage, Office of Communication, Stanford Medicine, to explain the proof-of-principle study published in PNAS by Phillip Cleves et al. (2018).
  6. Use this brief communication to explain how this work could allow researchers to use the CRISPR-Cas9 gene editing tool to identify and knock-out the coral genes responsible for coral bleaching due to ocean acidification.
  7. Discuss how this technique can thus be useful for coral conservation by building climate-resilient corals.

Suggested questions/assignments for learning evaluation :

  1. What is the function of CRISPR in bacteria? Describe the main components of the CRISPR-Cas9 system.
  2. Describe the two main DNA repair mechanisms in a cell.
  3. Explain how the CRISPR gene editing technique exploits the cell’s DNA repair system to introduce targeted mutations.
  4. How can CRISPR gene editing help plant breeding programs to adapt to the effects of climate change? Elaborate using a suitable example.
  5. Discuss the use of CRISPR technology as a climate adaptation strategy to conserve coral reefs.

The tools in this lesson plan will enable students to:

  1. describe the CRISPR-Cas9 pathway of adaptive immunity in bacteria
  2. learn about DNA repair mechanisms in cells
  3. understand CRISPR gene editing technique to achieve targeted mutations in cells
  4. use of CRISPR gene editing in living organisms as a climate adaptation strategy

If you or your students would like to explore the topic further, these additional resources will be useful.


George Q. Daley is dean of HMS, the Caroline Shields Walker Professor of Medicine, and a leader in stem cell science and cancer biology. As a spokesperson for the organizing committee of the Second International Summit on Human Genome Editing, he responded swiftly to He’s announcement in Hong Kong. Echoing those remarks, he said:

“It’s time to formulate what a clinical path to translation might look like so that we can talk about it. That does not mean that we’re ready to go into the clinic — we are not. We need to specify what the hurdles would be if one were to move forward responsibly and ethically. If you can’t surmount those hurdles, you don’t move forward.

“There are stark distinctions between editing genes in an embryo to prevent a baby from being born with sickle cell anemia and editing genes to alter the appearance or intelligence of future generations. There is a whole spectrum of considerations to be debated. The prospect includes an ultimate decision that we not go forward, that we decide that the benefits do not outweigh the costs.”

Asked how to prevent experiments like He’s while preserving academic freedom, Daley replied:

“For the past 15 years, I have been involved in efforts to establish international standards of professional conduct for stem cell research and its clinical translation, knowing full well that there could be — and has been — a growing number of independent practitioners directly marketing unproven interventions to vulnerable patients through the internet. We advocated so strongly for professional standards in an attempt to ward off the risks of an unregulated industry. Though imperfect, our efforts to encourage a common set of professional practices have been influential.

“You can’t control rogue scientists in any field. But with strongly defined guidelines for responsible professional conduct in place, such ethical violations like those of Dr. He should remain a backwater, because most practitioners will adhere to generally accepted norms. Scientists have a responsibility to come together to articulate professional standards and live by them. One has to raise the bar very high to define what the standards of safety and efficacy are, and what kind of oversight and independent judgment would be required for any approval.

“We have called for an ongoing international forum on human genome editing, and that could take many shapes. We’ve suggested that the national academies of more countries come together — the National Academy of Sciences in the U.S. and the Royal Society in the U.K. are very active here — because these are the groups most likely to have the expertise to convene these kinds of discussions and keep them going.”


Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the three genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4.

CRISPR Accelerates Science

Neuroscientist Steven Siegelbaum, PhD, has spent decades digging into the mechanisms of HCN1, a gene that serves as an electrical pacemaker within the human cortex, the part of the brain responsible for higher thought processes. In recent years, genome-wide association studies have implicated HCN1 mutations in forms of infantile and pediatric epilepsy that cannot be explained by a head injury, infection, metabolic disorder, or other clinical evidence. In some cases, seizures are so severe they lead to progressive brain dysfunction and developmental delays.

To develop the mouse models that could reveal how those mutations wreak such havoc, Siegelbaum and associate research scientist Bina Santoro, PhD, a longtime lead investigator in the Siegelbaum lab’s HCN1 research, turned to CRISPR. “It’s very fast, it’s comparatively cheaper than the traditional way of introducing point mutations, and there are a lot of these mutations in human patients that affect different parts of the HCN1 gene,” says Santoro. “We wanted to generate not just one mouse line, but a collection of mutations in the HCN1 gene, which are also present in human patients, to see the extent to which the mice reproduce the human condition.”

Using support from a Columbia Precision Medicine Initiative program and expertise in the Columbia transgenic mouse shared resource, Siegelbaum and Santoro have already developed four lines of mice with HCN1 mutations and seizure disorders and begun analyzing the morphology of their brains for preliminary clues about how the mutations affects brain anatomy and biochemistry. “In the best case, you save a year with CRISPR, maybe 12 to 18 months, depending on how lucky you are with the technique,” says Siegelbaum. “The general proof of principle, that these mutations are causing the seizures, will happen pretty soon.”

Deeper understanding—about the mechanisms by which proteins altered by the mutation affect electrical activity in the brain—will take considerably longer. “By using CRISPR we know that this one mutation to HCN1 is the only one in our experimental mice,” he explains. “And if they also develop seizures, that’s strong evidence that the mutation is a cause of the disease, not just associated. That’s our goal: We want to demonstrate that it’s the HCN1 mutations in the patients that are causing the disease.”

By simultaneously exploring multiple HCN1 variants and their role in seizures, Siegelbaum and Santoro also hope to gain insights into a basic conundrum about epilepsy, that seizure disorders take myriad forms and the drugs that can ameliorate symptoms in some patients aggravate the condition in others. “If in the mouse we can tie different mutations to different kinds of epilepsy,” says Santoro, “then we can see which mutations respond better to which drugs, or which drugs exacerbate which forms of the disease.”

Like Siegelbaum and Santoro, Lorraine Clark, PhD, assistant medical director of the Laboratory of Personalized Genomic Medicine, mixes genome-wide association studies, basic biochemistry and functional studies, and mouse models to reveal how gene variants affect brain function. Her research focuses on such neurodegenerative diseases as Parkinson’s and essential tremor.

Scientists already know that p.E326K, a specific variant of the glucocerebrosidase (GBA) gene, has been implicated in the severity of Gaucher disease and is one of the most common risk factors for Parkinson’s disease and dementia with Lewy bodies. Research suggests that the problem common to all three conditions has to do with how GBA encodes for the enzymes vital to the function of lysosomes, the organelles responsible for cellular digestion and waste removal. But scientists do not understand the specific mechanisms by which p.E326K disrupts enzyme production. Without that crucial insight, targeted therapies to ameliorate symptoms remain out of reach.

To learn more about how p.E326K alters lysosomal function, Clark is combining an award from the Columbia Precision Medicine Initiative with an R03 award from the NIH to generate a mouse model that has the gene variant so she can characterize the resulting brain pathology. “CRISPR is cost-effective, convenient, and easy to use,” says Clark. “Determining the disease mechanism associated with p.E326K may open up new therapeutic targets and could have a major impact on treatment of Parkinson’s disease and dementia with Lewy bodies.”

CRISPR Gene Editing Therapy Is Promising in New Mixed Model

A CRISPR-Cas9-based gene editing therapy promoted the production of a smaller but functional version of the dystrophin protein in a new mixed mouse model that uses muscle cells derived from Duchenne muscular dystrophy (DMD) patients.

The therapy is designed to delete a DMD gene region that is commonly mutated in people with DMD.

This is one of the first studies to prove the efficacy of this type of gene editing therapy in adult cells within a living organism, as opposed to patient cells grown in a lab dish. According to researchers, this type of data is important to support the evaluation of this gene-editing strategy in patients.

This new mouse model may be useful to test different kinds of investigational therapies and provide additional information on the potential application of gene editing or gene therapy for DMD, the investigators added.

DMD is caused by the loss of dystrophin, a key protein for muscle strength, due to mutations in the DMD gene. The most common Duchenne-causing mutations involve the deletion of one or more of its 79 exons — sections of genetic information needed to make proteins.

This deletion breaks the ability of the remaining exons to link together properly in an intermediate process of protein production, impairing the generation of a functional or properly working dystrophin protein.

To treat this, several therapeutic strategies have been developed to skip or permanently delete one or more DMD exons. The goal is to restore proper alignment of the remaining exons and allow the production of a working — although usually smaller — protein.

Originally discovered in bacteria as a defense mechanism, the CRISPR-Cas9 system allows researchers to edit parts of the genome by adding, removing, or changing specific sections of the DNA sequence.

Previous studies have shown that CRISPR-Cas9-mediated corrections of the DMD gene are highly effective at increasing the levels of functional dystrophin in patient-derived cells grown in the lab, and in mouse and dog models of DMD.

However, “an important step for the clinical translation of this gene-editing strategy is to demonstrate its efficacy and safety in human muscle fibers in vivo [in living animals],” the researchers wrote.

To fill this knowledge gap, a team from China created a new DMD mouse model harboring patient-derived muscle cells, called PDX DMD, and evaluated the effects of three different CRISPR-Cas9-based gene editing therapies in this model.

The first strategy was designed to promote the deletion of DMD exons 45–55, which account for about 63% of DMD-causing mutations, while the second set of deleted exons, 46–54, was thought to result in a potentially better working dystrophin protein. The last strategy targeted a specific region of exon 50 to restore the alignment of the remaining exons.

CRISPR gene editing components were delivered to cells through a modified version of an adeno-associated virus, often used in gene therapy.

The results showed that the first and second strategies, involving large deletions of the DMD gene, were highly effective at restoring dystrophin production. The researchers injected the gene therapy directly into the mice’s leg muscles that contained patient-derived muscle fibers.

Notably, dystrophin was detected in more than 10% of patient-derived muscle fibers in mice treated with either one of such strategies, while the third strategy was “inefficient in restoring dystrophin,” the researchers wrote.

Suggesting that the restored dystrophin was functional, both dystrophin and beta-dystroglycan — a molecule that forms a complex with dystrophin to provide structure to muscle cells — were found at their correct location in all rescued human muscle fibers.

The team also found that a potentially more specific CRISPR system, called CRISPR-Cas12a, was as effective as CRISPR-Cas9 at increasing the levels of a functional, but shorter, version of the dystrophin protein when designed to delete exons 46–54.

As such, the CRISPR-Cas12a system may be an alternative method for future gene editing therapy in DMD, the researchers noted.

“This study provides evidence for the efficacy of in vivo genome editing to correct disruptive mutations and restore dystrophin expression and function in DMD patient-derived muscle fibers,” they wrote.

Also of note, the large deletion strategies appeared to be more effective at restoring dystrophin and “might meet the requirements of clinical efficacy,” they added.

In addition, “the PDX DMD mouse model can be used to screen potentially effective strategies for clinical application,” the team wrote.

“The continued studies of gene-editing strategies, gene delivery approach, toxicology, and immunology in large animals will provide further insights into the potential application of gene therapy for DMD,” the researchers concluded.

CRISPR used to genetically edit coral

In a proof-of-principle study, Stanford scientists and their colleagues used the CRISPR-Cas9 gene-editing system to modify genes in coral, suggesting that the tool could one day aid conservation efforts.

Acropora millepora coral at the Australian Institute of Marine Science.
Phillip Cleves

Coral reefs on the precipice of collapse may get a conservation boost from the gene-editing tool known as CRISPR, according to researchers at the Stanford University School of Medicine and their collaborators.

The scientists found, for what appears to be the first time, definitive evidence that the CRISPR-Cas9 gene-editing tool could be a potent resource for coral biologists. Phillip Cleves, PhD, a postdoctoral scholar at Stanford, is a geneticist whose efforts to delineate gene function in animals resides squarely within the marine invertebrate realm — namely, corals.

“Up until now, there hasn’t been a way to ask whether a gene whose expression correlates with coral survival actually plays a causative role,” Cleves said. “There’s been no method to modify genes in coral and then ask what the consequences are.”

The study was published online April 23 in the Proceedings of the National Academy of Sciences. Cleves is the lead author. John Pringle, PhD, professor of genetics at Stanford, and Mikhail Matz, PhD, associate professor of integrative biology at the University of Texas-Austin, share senior authorship.

The damage of coral bleaching

In the late 1990s, the ocean’s coral reefs experienced the first big wave of something called coral bleaching, a bleak event in which ocean conditions — most prominently increasing temperatures — kill off or “bleach” parts of the reef, turning once-vibrant colors bland and damaging the entire reef ecosystem.

Researchers collect bundles of egg and sperm released as the coral colony spawns.
Phillip Cleves

Cleves’ work, conducted in collaboration with researchers at UT-Austin and the Australian Institute of Marine Science, sprouted from a conversation at an international coral meeting that aimed to concretely understand the genes behind coral survival. Are there some genes that render corals more resilient to spikes in ocean temperatures? Or perhaps a gene that helps establish new coral colonies? Scientists had hypothesized answers to these questions, but to truly know, Cleves wanted to create a technique that could allow coral biologists to answer such questions more rigorously.

“We want to use CRISPR-Cas9 with the express interest to start understanding what genes are critical to coral biology,” Cleves said.

CRISPR is a fast, effective tool that can be used to target and modify DNA sequences. “Breaking” genes to reveal the effects on the organism is a concept that’s been the linchpin of decades of molecular biology. Now, CRISPR is helping speed up the process in many diverse animal models, but applying it to corals (don’t be fooled — corals are animals, not plants) has proven tricky due in part to their infrequent reproduction. And until Cleves and his collaborators conducted this research, the use of the gene-editing tool had never been reported in corals.

“We hope that future experiments using CRISPR-Cas9 will help us develop a better understanding of basic coral biology that we then can apply to predict — and perhaps ameliorate — what’s going to happen in the future due to a changing climate,” Cleves said.

Spawning by moonlight

Corals pose a bit of a problem when it comes to CRISPR because of their spawning cycles. Most corals, including the Acropora millepora that was the focus of the study, breed only once or twice a year, during October and November in the Great Barrier Reef, cued by the rise of a full moon. During this fleeting window, corals release their sex cells into the ocean. When the eggs and sperm meet, they form zygotes, or fertilized single cells. During the narrow time window before these cells begin to divide, a researcher can introduce CRISPR by injecting a mixture of reagents into these zygotes to induce precise mutations in the coral DNA.

Retrieving the zygotes is quite a logistical challenge, Cleves acknowledged. Fortunately, his collaborators in Australia have the timing down pat they can predict when the moon spawn will occur within a couple of days, allowing them to take coral samples from the reef to gather zygotes for experimentation.

Cleves traveled to Australia to begin experimenting with CRISPR, targeting three coral genes: red fluorescent protein, green fluorescent protein and fibroblast growth factor 1a, a gene that is thought to help regulate new coral colonization.

Using CRISPR, the scientists made a type of genetic tweak that knocked out the genes, rendering them incapable of functioning. In the case of the red and green fluorescent proteins, determining if CRISPR worked would be easy — like seeing lights switch off. Or so they hoped. However, it turns out that there are multiple copies of red and green fluorescent-protein genes. So knocking out one copy didn’t put a stop to the glow altogether.

“Although we are not sure we saw convincing loss of fluorescence, DNA sequencing showed us that we were able to molecularly target both the red and the green fluorescent protein genes,” Cleves said. This showed the researchers that, in one go, CRISPR could successfully alter multiple genes if the two were similar enough — a boon to genetic manipulation, as genes are often duplicated during evolution.

As for the third gene, fibroblast growth factor 1a, which only has one gene copy, post-CRISPR sequencing showed success: in some embryos, the gene was largely mutated, suggesting that CRISPR will work well to modify single-copy coral genes.

Cleves said the ultimate goal is not to engineer a genetically resilient super-coral that could populate the ocean — such a feat is currently implausible and would raise significant ethical questions. “Right now, what we really want to do is figure out the basic mechanisms of how coral works and use that to inform conservation efforts in the future,” he said. “Maybe there are natural gene variants in coral that bolster their ability to survive in warmer waters we’d want to know that.”

‘An all-hands-on-deck moment’

Although the current work is a proof-of-principle study, now Cleves and others are beginning to tinker with genes that are more ecologically pertinent. And he hopes that others do the same.

“I want this paper to provide an early blueprint of the types of genetic manipulations that scientists can start doing with corals,” Cleves said. In the next few years, he hopes to see other groups knocking out coral genes potentially involved in bleaching, skeletal growth or the critical symbiosis with the algae that provide most of the corals’ energy.

Today, as much as 27 percent of the global reef ecosystem has been lost to a combination of climate change and human activities — and Cleves is feeling the urgency.

“This is an all-hands-on-deck moment,” he said. “If we can start classifying what genes are important, then we can get an idea of what we can do to help conservation, or even just to predict what's going to happen in the future. And I think that makes this a really exciting time to be a basic biologist looking at the genetics of coral.”

The research was funded by the Simons Foundation, the National Science Foundation and the Australian Institute of Marine Science.

Watch the video: What you need to know about CRISPR. Ellen Jorgensen (August 2022).