CRISPR in Medicine

Scientific Reviewer Fyodor Urnov

Introduction How can CRISPR tools treat – or prevent – disease?

You may have heard that CRISPR tools can alter disease-causing genes and may be able to treat a variety of disorders. This is true, but CRISPR tools may be able to treat or prevents diseases in many other ways as well! In this section, we dive into the many different ways doctors and researchers can use CRISPR tools to improve human health. Please note that many of the treatments discussed in this section are theoretical and/or currently in development. 

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Origins of genetic disease

Genetic disease, monogenic disease, polygenic disease

CRISPR tools help us study mono- and polygenic diseases

DNA encodes all the proteins that make up our cells. Certain DNA sequence variants encode malfunctioning proteins, or are "broken" so they don't encode a protein at all. These have the potential to cause genetic diseases. There are two main types of genetic disease:

  • Single gene disorders (monogenic diseases) - These are rare diseases caused by a DNA variant in a single gene or region. Researchers use patient data to identify such variants. These variants are tedious to recreate in lab and test with conventional techniques.
  • Multi-gene disorders (polygenic diseases) - These more common diseases are caused by combinations of DNA variants. Any one of these variants won’t cause disease on its own, but in combination, they can. Environmental and lifestyle factors can play a major role in the development of multi-gene disorders as well. Because of their complex etiology, multi-gene disorders are more difficult to study than single gene disorders.

CRISPR tools make it much easier to study both types of genetic disease.

Genetic diseases

DNA variants lead to disease because they alter cellular functions. Some DNA variants cause very important cellular parts to malfunction. Other variants encode completely non-functional parts, or are "broken" in a way that stops a protein from being made at all. Some DNA variants are in noncoding regions of DNA that have important functions like controlling gene expression. With such intense impacts, these variants can, on their own, lead to defective cells. When this happens, it results in a single gene disorder.

Think of a cell like a sports team. A DNA variant that causes a monogenic disease is like an injured star player or a benched mediocre player.

Other DNA variants lead to dysfunctions that aren’t quite so impactful. These may encode proteins with reduced function. They may also encode nonfunctional versions of less important proteins, or stop a non-essential protein from being made altogether, or impact gene regulation. A cell can compensate for each dysfunction individually, but together the small individual dysfunctions add up to defective cells. When this happens, it results in a multi-gene disorder.

Think about the sports team again. DNA variants that cause multi-gene disorders are like the entire team having a bad game. Normally other team members can compensate if one person is having a bad day, but the team will lose if everyone underperforms. In multi-gene disorders, many genes underperform to cause disease.

It is important to note that disease is often a product of an interaction between DNA variants and lifestyle, life experience, and/or the environment. Sometimes DNA variants encode proteins that malfunction only under certain conditions. Other times, the combination of life style, experience, or environmental factors combine with DNA variants to lead to disease. For example, some people may have a DNA variant that makes them more likely to develop asthma. But, asthma only occurs when people with that variant live in areas with moderate or high air pollution. In this way, the environment has a big impact on whether DNA variants lead to disease.

Further, some health issues are almost entirely due to life style, life experiences, and environment. For example, back problems can result from an occupation involving heaving lifting. There might be some gene variants that make people more or less prone to back issues, but these will generally have little impact compared to a lifetime of heavy lifting.

Using CRISPR genome-editing tools to study genetic diseases

CRISPR genome-editing tools facilitate single and multi-gene disorder research. The first step of this work is for researchers to determine if suspected DNA variants actually cause disease. To do so, they add the variants to healthy cells and remove them from diseased cells. If the variants cause disease, the healthy cells get sick and diseased cells get better.

For single-gene disorders, researchers need to add or subtract just one DNA variant. CRISPR genome-editing tools make this process easier and faster. Using these tools, researchers can test variants in many cells under many conditions. They can quickly learn a lot about these diseases and how they impact cellular function.

CRISPR tools are even more impactful in multi-gene disorders. With multi-gene disorders, researchers must study many DNA variants at once. This was not workable before CRISPR genome editing. CRISPR tools make polygenic disease research possible for two reasons:

  1. It is easy to direct CRISPR genome-editing tools to many sites - Researchers simply use many guide RNAs at the same time to direct CRISPR tools to many DNA sequences simultaneously. Before CRISPR tools were available, researchers had to make entire new tools to edit each new site. This was very time consuming and impractical for all the sites in multi-gene disorders.
  2. CRISPR genome-editing components are small and easy to deliver - Old genome-editing tools were very large and it was hard to deliver more than one at once. The guide RNAs used in CRISPR genome-editing are small. So, researchers can deliver many at once and theoretically edit all the sites in a multi-gene disorder.

With CRISPR tools, researchers are learning much more about DNA variants and disease. Using this information, they will create new treatments. Some will rely on using the knowledge gained from CRISPR-based research to develop traditional small molecule drugs, antibody-based treatments, and more. Some will use genome editing to directly change disease-causing sequences to healthy versions. 

Disease modeling

How scientists study diseases in the lab

Xenograft, chimera, disease model

Learning how to treat diseases in human-relevant models

To develop new treatments for disease, researchers need ways to test their safety and efficacy before testing them in clinical trials with human patient-volunteers. This is where disease models step in.

Disease models simulate the important characteristics of human diseases. These models can take many forms, from cells in petri dishes to whole animals.

Using CRISPR tools to create disease models

CRISPR tools help researchers create disease models in two main ways:

  1. By enabling researchers to verify that candidate disease-causing DNA sequence variants do indeed cause disease
  2. By causing cells or animals to take on disease-like characteristics

For example, pretend you are a researcher studying diabetes. You might identify potential diabetes-causing DNA sequence variants by analyzing DNA from diabetic individuals. To verify that these variants cause disease, you could use CRISPR tools to insert them into pancreas cells grown in a dish. You could then test whether these cells take on characteristics associated with diabetes. For instance, you might test if the cells produce insulin.

Once verified, researchers use DNA sequence variants to simulate diseases in human-relevant models. First they choose appropriate platforms for their disease models. A platform could be cells in a dish, cells grown together into tissues or organ-like structures, or even whole organisms.

The particular characteristics of a disease and the particular experiments need to be considered carefully when choosing a model. For a disease like diabetes that affects the whole body, sometimes a whole organism would be a better choice. A wise choice would be an organism with a pancreas because many of the effects of diabetes operate through the pancreas. A mouse could be a good option.

Simulating diseases with CRISPR tools

After carefully choosing a platform, the next step is to simulate the disease in the platform. There are a variety of ways to do this:

  • Use CRISPR tools to edit DNA sequences in specific cells - For example, researchers could insert diabetes-causing DNA variants into mouse pancreatic cells.

  • Use CRISPR tools to edit DNA sequences in whole organisms - Here researchers insert disease-causing DNA sequence variants into egg/sperm cells or embryo cells. These cells later give rise to organisms with disease-causing DNA variants in every cell.
  • Use xenografts - A xenograft consists of tissue transferred from one species to another. Xenografts enable researchers to study diseased human cells in non-humans. Xenografts can come from a variety of sources, including patients or genome-edited cells grown in the lab. For example, researchers can transfer human tumors to mice. Later they can study how the tumors affect the mice. They can also test how drugs delivered to the mice impact the tumors.
  • Create chimeras - Chimeras are organisms composed of mixtures of cells with different DNA sequences. They might even contain cells from different species. Chimeras can be useful for studying human diseases in non-humans. For example, researchers can create pigs containing human cells in their organs. Such “humanized” organs might better simulate human disease. Researchers use complex genome editing and stem cell-based techniques to create chimeras.

Researchers don’t have to use genome editing to create disease models. For example, CRISPR tools that turn up and down genes can also cause disease-like effects. Even chemical treatments can cause disease-like symptoms. Nonetheless, diseases caused by DNA sequence variants are often best modeled using the same variants. 

With an appropriate model in hand, researchers can learn a lot about disease biology. They can use models to learn how diseases impact particular organs or tissues. They can also use models to test new treatments. If a treatment is effective in a model, researchers might later test it in humans. Because CRISPR tools make it much easier to make disease models, they help researchers get new treatments to the clinic more quickly!

Genome-editing therapies

How scientists are developing CRISPR-based therapies to treat diseases

Genome editing, cell therapy, in situ genome editing, off-target edits, germline editing, somatic cell, germ cell

Adding or changing DNA to treat disease

In genome-editing therapies, scientists deliver helpful DNA sequences to cells. These DNA sequences can play a variety of roles. They might make small edits to DNA to revert to a healthy variant of the DNA sequence or turn a gene "on" or "off." They might replace sections of a DNA sequences that cause disease. They might also give cells new abilities. CRISPR tools make gene therapy easier, but are not the only way to do it.

This article divides genome-editing therapies into three groups. In order of practicality, they are:

  • Cell therapies* - These use lab-modified cells to treat disease.
  • In situ genome-editing therapies* - These use genome-editing tools to fix diseased cells in patients.*Both cell and in situ therapies use 'somatic' genome editing. That is, they alter adult cells that cannot give rise to offspring.
  • Germline therapies - These use genome editing to alter DNA in cells that will form entire organisms (think eggs and sperm). Thus, all cells that make up the resulting organisms have altered DNA. Their future offspring will inherit their altered DNA.

These genomic therapies come with escalating levels of ethical and technical difficulties. The first two types of are already reaching the clinic. Germline therapy requires more research, public discussion, and oversight before it reaches the clinic (if at all).

Cell therapy

Cell therapies use modified cells to treat disease. Generally these cells are isolated from a patient or an individual acting as a cell donor, and then genome edited and multiplied in the lab. After editing, doctors deliver these cells (back) to patients, often by IV infusion. Once in the body, the genome-edited cells fight a specific disease.

A CRISPR-based treatment for sickle cell disease, which is being studied in clinical trials, is a great examples of a cellular therapeutic. In sickle cell disease, a faulty form of the protein hemoglobin causes red blood cells to be misshapen, or "sickled." In the treatment, blood stem cells are harvested from an individual, edited in the lab, and then infused back into the patient, where they take up residence in the bone marrow and start making healthy, round red blood cells. 

Cell therapies are often more practical than the other forms of genome editing for many reasons:

  • The genome-editing process is easier - It's less technically challenging to edit cells in the lab than in the body. Scientists also usually have more cells to work with in the lab.
  • It's easier to edit the specific cell type - When genome-editing tools are in the body, there's a risk of them modifying cell types besides the kind being targeted. If cells are isolated from the patient and modified outside of the body, there is very little risk of editing the wrong cell types.
  • Modified cells can be easy to deliver to patients - These are generally infused into the bloodstream or delivered to a particular organ.
  • There is little or no risk of passing on the modified cells to future generations - In this sense, cellular therapeutics are like standard drugs. They only directly impact the individuals they treat. So, they raise fewer ethical issues than the other types of genome editing.

Future cell therapies may cure a variety of diseases. Some scientists are working to modify liver cells to cure blood diseases like hemophilia. Others are working on ways to create pancreas cells to cure diabetes. Still others are working on making immune cells resistant to HIV infection. And researchers are finding new ways to isolate and modify cells all the time. 

In situ genome editing

In situ means “in the original place.” In situ therapies treat diseases at their source in defective cells. They use genome editing to alter disease-causing DNA variants in the body.

In situ genome editing is generally more difficult than cell therapy. It is harder to deliver genome-editing tools  directly to cells in the human body than to isolated cells in the lab. Most cells are not as easily accessible as those in the eye. Think about how difficult it could be to deliver genome-editing tools to widely distributed cells like muscle cells. Many diseases involve such widely distributed cells.

Even when target cells are accessible, in situ genome editing is challenging because it must be very precise. If genome-editing tools act in the wrong cells, there could be negative health consequences. Researchers can achieve some precision using special viruses. These viruses are themselves modified so they no longer cause disease, but still have their native ability to get into specific kinds of cells. Genome-editing tools can be packaged inside the viruses, who carry them into specific cells. There is always some risk because viruses aren’t 100% accurate. Researchers also don’t have viruses to target each type of cells.

Although unlikely, in situ genome editing could also result in germline editing, causing future generations to inherit DNA alterations made by the genome-editing components. This would happen if genome-editing tools were accidentally delivered to egg or sperm cells. So, researchers must be very careful when developing in situ gene therapies to minimize the risk of these off-target edits

Germline editing

Germline editing is highly controversial. It uses genome-editing tools to alter sperm, eggs, or embryos – cells that give rise to whole organisms. As a result, these organisms pass on the changes in their DNA to their offspring generations. In other words, germline editing impacts future generations and not just individual patients. For this reason, it brings up many more ethical questions than cell or in situ editing and is prohibited by many countries. There is currently no country that allows heritable germline genome editing, meaning, changes that would be passed on to generations beyond the initial edited organism. 

However, germline editing is worth thinking about because certain diseases could most effectively be treated effectively with germline editing. Diseases caused by DNA variants that alter many cells in the body are a good example. Their health impacts are too broad for cell therapies and in situ gene therapy cannot reach all the defective cells. Germline therapy is also the most effective option for diseases that have effects very early in development. These diseases might be severely disabling or fatal before birth or in infant or toddler years, so preventing them from developing at all is the most effective approach.

Despite these potential uses, there are many reasons germline editing is controversial:

  • Scientists don’t know how to do germline editing safely yet - It’s unclear whether current genome-editing techniques are accurate enough for germline therapy. 'Off-target' germline effects or unwanted 'on-target' effects would result in modifications throughout the entire treated organism, potentially resulting in drastic health consequences. 

    Off-target editing and unwanted on-target effects are possible in the other types of gene therapy, but the risk is less. Scientists edit a limited number of cells in cell and in situ treatments. This lowers their chances for drastic off-target consequences. In cell therapies, scientists can even test their cells for off-target editing. So, they can treat patients using only cells with the correct edits. It is not possible to confirm that an edited embryo has only wanted changes without destroying the embryo. 

  • Germline therapy has the potential to alter the human gene pool - Many people think that permanently altering human genetics is a bad idea. They are concerned that changing the gene pool might have unforeseen negative consequences on future generations. Bolstering this argument, some disease-causing DNA variants have health benefits in particular environments. As such, it may be unwise to take actions that could remove these variants from the gene pool.
  • Germline therapy could be used for enhancement as opposed to treatment - Here “enhancement” means trying to improve human genetics for reasons beyond treating or preventing disease or disability. For example, parents could use germline editing to give their children bigger muscles. While many human characteristics like intelligence are too complicated for this to be done in the short term and too shaped by environment to be realistic even in the long term, some kinds of enhancements are theoretically possible. Detractors believe that enhancement could exacerbate inequities between classes, races, geographies, and more. Enhancement might be possible to a lesser extent with the other types of genome editing, but those DNA changes wouldn't extend beyond individual patients.
  • Inherent lack of consent - Germline therapy “treats” germcells that will form fetuses, and ultimately, new individuals. However, these future individuals have no means to consent to the treatment. This is compounded by the fact that germline edits are heritable: as a result, lack of consent extends far beyond the treated individual and into future generations.

These issues are important and societies must consider them before using germline editing. It needs extensive and inclusive discussion, debate, consent, and regulation before it could be used ethically. For now, families with genetic risks have options including carrier screening, embryo screening, and prenatal diagnostics. 

Find the Official IGI Stance on Human Germline Editing here.

Delivering CRISPR therapies

Systemic delivery, local delivery, virus, lipid nanoparticle/LNP, "naked" CRISPR tool, ribonucleoprotein/RNP

Getting CRISPR tools into human cells for genome editing

CRISPR therapies can have profound impacts on disease. Their effectiveness depends on our ability to deliver CRISPR tools to cells. Physicians need ways to distribute CRISPR tools to specific cell types. They also need ways to get these tools across cellular barriers.

Researchers have come up with many ways to get CRISPR tools into cells. Methods for systemic delivery  involve broadly distributing CRISPR tools throughout the body, and the delivery tool (discussed in detail below) targets specific cells as it moves through the whole body system. Methods for local delivery get CRISPR tools directly to specific tissues or cells.

Which delivery method a physician uses depends on the specific therapy. With diseases that impact cells throughout the body, systemic delivery is very important.  For example, muscle wasting diseases like muscular dystrophy might need systemic delivery. With diseases that impact specific cells or tissues that can be easily accessed, local delivery may be the best option. For example, certain bladder conditions could benefit from local delivery to the bladder. Indeed, local delivery might be a better option in these cases because it can limit side effects and the risk of unwanted edits in other cell types.

We discuss three of the most popular methods for delivering CRISPR therapies below. These have varying levels of potential for systemic and local delivery. Each also has its own pros and cons. Researchers are actively working on improving all these methods and creating new ones.

1. Specialized viruses

Viruses naturally infect human cells. During this process they often release their DNA into the infected cells. Normally, this DNA encodes proteins that force cells to produce more copies of the virus. Ultimately, the viruses sometimes kill the infected cells.

Researchers have discovered ways to domesticate some viruses. The domesticated viruses no longer turn cells into virus-replicating factories. Instead, they carry genome-editing tools and release them into their target cells. Researchers can deliver DNA encoding CRISPR tools into specific cell types using these viruses. Adeno-associated viruses (AAV) are commonly used for this.

Viruses can deliver CRISPR tools either systemically or locally. Some viruses are great at systemic delivery. Some are more suited for local delivery. Researchers can even modify some viruses to make delivery more or less specific. They are very versatile.

Viruses do have some drawbacks. For example, some can cause powerful immune reactions. Others may insert their CRISPR-encoding DNA into random locations in the genome. Still others can only deliver small pieces of DNA. The first two drawbacks could result in severe health consequences for patients. The third puts limitations on the types of CRISPR tools that can be delivered. However, viruses have a long history in gene therapy and, more recently, in genome editing, so physicians know how to monitor and treat their side-effects.

Viruses can stick around in cells for weeks, months, or years. When viruses are used to deliver genome-editing components, it can result in the presence of genome-editing components for weeks, months, or years. This may be desirable in some cases, but also increases the risk of immune reactions to the virus or the CRISPR proteins, as well as the risk of off-target effects, as opposed to other methods which lead to transient expression of genome-editing components. 

2. Nanoparticles

Lipid nanoparticles are small, spherical structures made out of lipids (fats) and may also be referred to as LNPs.  Researchers have various ways to encapsulate CRISPR tools inside these structures. They easily pass through membranes and deposit their CRISPR cargoes into cells. Usually the CRISPR tools are encapsulated as mRNA or protein.

LNPs are easy to prepare and researchers can easily modify their structures. Some formulations might better penetrate certain cell types, pass through bodily barriers, or have more stability. Unlike viruses, nanoparticles don’t tend to provoke immune responses, so they may have fewer side effects and be more safe than viruses when administered to the body. 

LNPs can be used to local delivery. If injected into the bloodstream, these particles accumulate in the liver, making them great for systemic therapies that target the liver. However, they may have low effectiveness when delivered systemically for therapies targeting other cells in the body. Researchers are working on developing nanoparticles that are more versatile for systemic delivery to cells, tissues, or organs besides the liver.

LNPs lead to transient expression of genome-editing components. Within a week or so of administration, there will be no trace of the nanoparticles of genome-editing components. This decreases the risk of immune reactions to the CRISPR proteins, as well as the risk of off-target effects, relative to viral delivery.

3. Direct delivery of naked CRISPR tools

In the above methods, researchers encapsulate CRISPR tools before delivery. In some cases it makes sense to deliver non-encapsulated "naked" CRISPR tools. For example, cell therapies use modified cells to fight disease. These cells are usually modified in the lab and then injected or infused back into patients. In the lab, researchers can add the naked CRISPR tools directly to the growing cells. With the proper mixture of chemicals or electric pulses, these cells can be coaxed to take up the tools.

It’s also possible to modify naked CRISPR tools to make cells in the body more likely to take them up. These naked but disguised tools are like trojan horses, and cells will pull these disguised tools into their membranes. Once inside, the tools get to work editing DNA.

Currently it only makes sense to deliver naked CRISPR tools locally. Naked CRISPR tools can provoke immune responses. They're also not stable enough for systemic delivery. Nonetheless, researchers are actively coming up with ways to improve naked delivery. Like nanoparticles, naked CRISPR tools would be present only transiently. 

CRISPR delivery options and improvements to come

Researchers are working on ways to improve these delivery methods and develop new methods to get CRISPR tools to the right cells and reduce risks and side effects.


How CRISPR tools could be used to help replace human organs with animal organs

Xenograft, xenotransplant

Replacing human organs with animal organs

Every year, thousands of people die waiting for organ transplants because there are just not enough organs available. CRISPR genome-editing tools may be able to ameliorate this problem. Researchers hope to use these tools so we can replace human organs with animals organs. These xenografts could potentially save many human lives.

What is a xenograft?

Xenografts are tissues or organs transplanted from one species to another. Xenografts help scientists study human tissues. They can also treat human ailments. For example, doctors can replace human heart valves with pig heart valves. Doctors can also treat burn victims with pig or fish skin grafts.

Replacing whole human organs with xenografts is difficult. The human immune system has many ways to detect and reject non-human cells. Non-human organs can also carry zoonotic diseases which could put patients at risk.

Making xenografting more workable with CRISPR genome editing

Researchers can use CRISPR genome-editing tools to modify whole animals. Certain modifications could result in making animal organs more amenable to xenografting. Such modifications come in the following forms:

  • Modifications that delete viral DNA - For example, pigs have viruses hiding in their DNA. In xenografts, these viruses could infect human patients. Researchers have removed many of these viruses from pigs using CRISPR genome editing. So, organs from these pigs should be less dangerous to humans.
  • Modifications that make the human immune system less likely to attack the xenograft -  Some sequences of animal DNA encode proteins recognized by the immune system. Using genome editing, researchers can remove or modify these sequences. They can also use genome editing to give animals DNA sequences that calm down the immune system.
  • Modifications that “humanize” animal organs - These modifications are controversial. To make them, researchers use genome editing tools to put human DNA into animals. This DNA encodes cellular parts that make animal organs more human-like. In addition, researchers can replace cells in developing animals with human cells. Under the right conditions, the human cells will form entire organs in the animals. These essentially human organs should be easier to transplant.

Since the 1960s, there have been a handful or so of xenotransplantations done without success. In 2022, the first xenotransplantation aided by CRISPR was performed; the patient only survived for two months after the operation. Whether xenografts are adopted more broadly depends not just on improving the protocols, but also on opinions of xenografts by society at large. Societies must weigh the risks and benefits of xenografts, as well as concerns for animal welfare. 

Nucleic acid detection

How CRISPR proteins can be used to detect specific nucleic acid sequences

Cas, nucleic acid

Detecting disease-causing organisms with CRISPR

Some diagnostic tools detect disease-causing organisms. Doctors use them to discover why people are sick. Others use them to test food and drinking water for microbial contamination.

One way to detect disease-causing organisms is to identify their DNA. Unfortunately, many DNA identification methods are difficult to use. CRISPR DNA detectors may provide a simple alternative: they can be made to light up in the presence of specific DNA sequences.

How do CRISPR nucleic acid detectors work?

CRISPR-based DNA detectors have 2 key components:

  1. Cas proteins that cut apart RNA or DNA (nucleic acids) in a non-targeted way after cutting a user-specified DNA sequence.
  2. Special kinds of nucleic acids that glow when cut.

In DNA detectors, researchers combine these two components with DNA samples. If the user-specified DNA sequence is present, then the following happens:

  1. The Cas protein cuts the target DNA sequence.
  2. The Cas protein starts to cut apart nucleic acids in a non-targeted way.
  3. The Cas protein cuts apart the special nucleic acids.
  4. The special nucleic acids glow.

The glowing nucleic acids are easy to see. They effectively tell users that target DNA is present.

CRISPR-based DNA detectors are easy to target to new sequences. Researchers just swap out the guide molecules directing them to their targets. Thus, CRISPR DNA detectors can spot many organisms.

For example, a doctor could use CRISPR-based DNA detectors to diagnose disease. The doctor would create many DNA detectors. Each would glow upon detecting DNA from a different disease-causing organism. The doctor would then add patient samples (e.g. blood) to the detectors. Depending on which detector glowed as a result, the doctor would find out which organism made the patient sick. Then, the doctor could prescribe an appropriate treatment.

This is just one example of a CRISPR-based tool impacting health without genome editing. In general, CRISPR technologies are powerful because of their customizability and ease of use.  Hopefully we’ll continue to see more creative applications of CRISPR technologies in the future.

Safety concerns for CRISPR therapies

Off-target editing, immune responses to CRISPR proteins, and unintended effects

Making sure CRISPR tools do the right thing in the right place at the right time

Off-target effect, guide RNA, anti-CRISPR

CRISPR therapies have many uses, but all face some common safety risks. Below we discuss some of these risks and ways to mitigate them.

Off-target effects

CRISPR tools occasionally make changes at the wrong location in the DNA, usually sequences that are similar to the target DNA sequence. These are known as off-target effects.

Off-target effects could have have drastic consequences. For instance, they might change DNA sequences that control cell growth, making cells cancerous. Other unwanted DNA changes might prevent cells from carrying out important functions. For example, they might prevent pancreas cells from producing insulin.

There are many ways to reduce the risk of off-target effects. You may recall that guide RNAs (gRNAs) target CRISPR tools to specific DNA sequences. Researchers can test many gRNAs to find the most accurate ones. For instance, they can test their gRNAs and CRISPR tools on cells in the lab. Then they can examine the DNA sequences in these cells. If they find a high frequency of off-target effects, they will likely chose to redesign their gRNAs.

Researchers have also modified CRISPR tools to make them more accurate. With any given gRNA, these enhanced tools are less likely to edit the wrong DNA sequences.

Finally, turning off CRISPR tools can decrease the chances for off-target effects. To understand why, think of CRISPR tools like super accurate archers. With enough shots, even the most accurate archer will miss their target at some point. Similarly, CRISPR tools will edit the wrong DNA sequences if given enough time. Thus, off-switches limit the amount of time CRISPR tools are functional, giving them fewer chances to make mistakes.

Scientists use anti-CRISPRs – proteins that block CRISPR tools – to turn CRISPR "off." 

Provoking immune responses

While defending us, the immune system searches for non-human proteins or parts of them. Normally such proteins come from infectious microbes. If immune cells find these proteins, they sound alarms and activate immune responses.

CRISPR proteins come from bacteria and archaea and can activate immune responses in human patients. These immune responses might stop CRISPR therapies before they can help patients. Strong immune responses can also cause acute inflammation, which can make patients sicker.

Researchers overcome immune responses in a few ways. First, they can give patients immunosuppressive drugs. These lower immune responses and mitigate adverse reactions. Such drugs are widely used in clinical settings and are particularly important for things like organ transplants. They are very useful but, as a side effect, increase the potential for infections.

Researchers also hope to lower the potential for immune responses by altering the make-up of their CRISPR proteins. These altered proteins wouldn't recognized by the immune system, so they’re less likely to rouse immune responses.

Finally, researchers can turn on and off the production of CRISPR proteins. With these capabilities, researchers can produce CRISPR proteins only when and where they are needed. As a result, there will be fewer CRISPR proteins in the body. With less protein, immune responses are less likely and less severe when they do occur.

Unintended effects of genome editing

Occasionally CRISPR genome editing has effects that are difficult to anticipate or detect. At a technical level, the DNA cutting process required for genome editing can result in large scale rearrangements and changes to DNA that are difficult to see. These may cause problems for CRISPR therapies.

Even if everything goes smoothly, there can be unintended consequences from CRISPR genome editing. The “disease genes” altered in CRISPR therapies often have important functions in healthy people. “Disease genes” might even be beneficial under certain conditions. So, if we get rid of them with CRISPR therapies, there could be dangerous consequences. 
For example, researchers have used genome editing in an attempt to protect people from HIV infection. To do so, they altered a specific protein on patients' immune cells. HIV normally grabs onto this protein to get into immune cells, so doctors hope cells with this altered protein will resist HIV infection. However, this altered protein may also make patients more susceptible to other infections.
It seems simple to get rid of a “disease gene.” But it’s important to remember that biology is complex. A single gene may be important in many processes. Researchers must carefully study the impacts of genome editing through extensive clinical trials before they go mainstream. You can learn more about clinical trials here.

Safer CRISPR tools will result in more therapeutic impacts

Over time, researchers will get better at reducing the risks of CRISPR-based treatments. It is safe to assume that CRISPR therapies will have greater positive impacts on health over time, too.