Demystifying Gene Editing With CRISPR

Gene editing has received a great deal of media attention in recently due to its potential for advancing science and treating disease. In a short period of time, scientists have developed several powerful tools that are capable of introducing extremely precise genomic alterations. In particular, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), which was named Science’s 2015 Breakthrough of the Year and for which the developers received several prestigious awards, has been widely adopted by scientists due to its relative affordability, efficiency, and ease of use.1

Gene therapy is predicted eventually to benefit millions of patients by enabling shorter treatment regimens with longer-lasting curative benefits, or – extrapolating the potential of gene editing to its logical end –by simply “cutting out” genetic diseases. However, as with all medical advances, gene editing and gene therapies also raise questions about ethics, risks, affordability, and regulation.2

“The new editing technology is a revolution for science and for medicine,” Stuart Orkin, MD, associate chief of the division of hematology/oncology and chairman of the pediatric oncology department at Boston Children’s Hospital and David G. Nathan Professor of Pediatrics at Harvard Medical School, in Boston, Massachusetts, told ASH Clinical News. “The technology is being rapidly improved, and it is highly likely that it will be applied for several disorders in the near future, with positive effect.”

So, what is CRISPR/Cas9, and what do hematologists need to know about this technology as it transitions from the laboratory to the clinic? ASH Clinical News spoke with Dr. Orkin and other researchers specializing in CRISPR/Cas9 gene editing for answers.

“Today, we are
limited more by
our imagination
and by figuring out
the right question
than by the tools at

—Margaret Goodell, PhD

Gene Editing 101

Gene editing (also known as genome editing) refers to the alteration of DNA at specific locations in the genome.3 Several gene-editing technologies have been developed, all based on nucleases (enzymes that cleave nucleic acids) that are delivered to targeted cells, then recognize, bind, and cleave a target sequence of DNA.

Gene editing takes advantage of the cell’s own DNA repair mechanisms, which can result in many different molecular outcomes, such as:

  • non-homologous end joining, which reunites the broken ends of DNA and often results in small insertions or deletions, and can lead to gene disruption4
  • homology-driven repair (HDR), which aids in introducing novel DNA by exploiting donor DNA molecules that have homologous sequences surrounding the DNA break5

Meganucleases were the first targeted nucleases to be used for gene editing.4 More recently, gene editing has been revolutionized by  nuclease-based technologies with improved speed, cost, accuracy, and efficiency:3


Zinc-finger nucleases (ZFNs) are engineered DNA-binding proteins that facilitate targeted genome editing by creating double-strand breaks in DNA at specified locations. ZFNs consist of a FokI nuclease domain and three to six DNA-binding zinc-finger domains. Because the FokI nuclease functions as a dimer, a pair of ZFNs is engineered to bind nine to 18 base pairs of DNA on either side of the target sequence. Like meganucleases, ZFNs require complicated engineering for each new target DNA sequence.4,5


Transcription activator-like effector nucleases (TALENs) are also fusions of a FokI nuclease domain and a DNA-binding domain (transcription activator-like [TAL] proteins). When two TALENs bind and meet, the FokI domains create a double-strand break that can “turn off” a gene or can be used to insert DNA. One advantage of TALENs over ZFNs is their more straightforward, modular engineering: Each TAL binds a single DNA base, so they can be arranged in any order to create novel DNA-binding domains. Like ZFNs, specificity is increased due to the requirement for dimerization of FokI. Recently, smaller hybrid megaTALs have been created from meganuclease plus TAL repeats.4,5


CRISPR/Cas9 is the most recent gene editing tool to be added to the repertoire. It is adapted from a naturally occurring genome editing system in which bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to “remember” the invading viruses (or closely related ones). If they invade again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses’ DNA. The bacteria use Cas9 or a similar enzyme to specifically cleave the viruses’ DNA, which disables the virus.3

In the CRISPR/Cas9 technology most often used for gene editing, a single guide RNA (sgRNA) directs Cas9 to cleave at a specific site – essentially cutting out and shutting off the targeted gene.

Scientists can create a new sgRNA for any genomic target, and the Cas9 nuclease cutting tool can be used with multiple sgRNAs to make multiple changes simultaneously. CRISPR/Cas9’s use of RNA as a reagent offers a major advantage over ZFNs and TALENs, which require complicated and expensive protein engineering for each new target.4,5

“CRISPR is easy, efficient, and relatively inexpensive. This is such an accessible technology that we have an entire new toolkit in the community,” said Margaret Goodell, PhD, a professor at Baylor College of Medicine and director of the Stem Cell and Regenerative Medicine Center in Houston, Texas. “The pace of experiments and the types of experiments that we can now do has fundamentally changed. Today, we are limited more by our imagination and by figuring out the right question than by the tools at hand.”

“There are no ethical concerns unless one talks about germline editing, which is not generally thought of at this time for hematologic disease.”

—Stuart Orkin, MD

Ethical Considerations of Gene Therapy

Gene therapy refers to the modification of a dysfunctional gene to treat or cure disease. Today’s gene-editing technologies can be used for gene therapies, which fall into the following two categories:3

  • somatic therapies alter the DNA in non-reproductive cells so changes affect only the person receiving therapy
  • germline therapies alter the DNA in reproductive cells so changes can be passed down to future generations

Somatic gene therapy products are classified as “biological products” and are regulated by the U.S. Food and Drug Administration’s (FDA) Center for Biologics Evaluation and Research. Proposals for gene therapy clinical trials funded by the National Institutes of Health (NIH) are reviewed by its Recombinant DNA Advisory Committee (RAC), which collaborates with the FDA.6

The FDA is prohibited from reviewing proposals for studies that use germline editing to alter human embryos. Should this restriction expire, the National Academies of Sciences, Engineering, and Medicine proposed stringent criteria for adopting germline editing, recommending that germline editing research trials be permitted “only for compelling purposes of treating or preventing serious disease or disabilities, and only if there is a stringent oversight system.”7 However, many scientists believe that any germline editing using current technologies would be dangerous and unethical.8

As far as CRISPR/Cas9 is concerned, Dr. Orkin noted, “There are no ethical concerns unless one talks about germline editing, which is not generally thought of at this time for hematologic disease.”

The Role of CRISPR/Cas9 in Hematology

To date, no commercialized CRISPR/Cas9 products have begun clinical trials in the U.S. “That being said, there are multiple programs in development – both in academia and industry – heading in that direction,” said Matthew Porteus, MD, PhD, associate professor of pediatrics at Stanford University in California, and founder of CRISPR Therapeutics. “We are likely to see clinical trials in 2018 and 2019.” At Stanford, Dr. Porteus’s group is moving toward a clinical trial for CRISPR/Cas9 gene editing in sickle cell disease (SCD).9

“It is rather remarkable that the system was first described as useful in mammalian cells in 2013, and we learned how to use the system efficiently in clinically relevant human cells in 2015,” he added. “The pace to the clinic is amazingly fast given the careful scientific and regulatory work that needs to be done before one can give genetically engineered cells to patients.”

Enhancing Cellular Immunotherapy

To date, the FDA has approved two gene therapies, both autologous chimeric antigen receptor (CAR) T-cell immunotherapies for treating types of leukemia and lymphoma.10,11 Therapy involves collection of the patient’s own T cells, genetic modification to express a CAR that targets tumor cells, and infusion of modified T cells back into the patient’s system.12

These CAR-modified T cells are made using viral delivery systems, which randomly insert the CAR gene into the T-cell genome and may result in unwanted genetic effects. Scientists are now using CRISPR/Cas9 to generate CAR T cells and have found that controlling where CAR integrates can enhance their potency.13

CRISPR/Cas9 gene editing also is being investigated as a tool to enhance CAR T-cell function by disabling genes that encode inhibitory receptors or signaling molecules, such as programmed cell death protein 1 (PD1).12 The first CRISPR/Cas9 clinical trial, initiated in China in 2016, is doing just that.14

In the U.S., a proposed CRISPR/Cas9 clinical trial has already been approved by RAC, but still needs approval from the FDA. This clinical trial will focus on the safety of CRISPR/Cas9-modified T cells for the treatment of myeloma, sarcoma, and melanoma. CRISPR/Cas9 will be used to knock out PD1 as well as the endogenous T-cell receptor.15

“Such gene editing promises to improve the potency and specificity of lymphocyte products and may even enable production of cell therapies from universal donors,” Daniel Bauer, MD, PhD, from the department of pediatric hematology/oncology at Boston Children’s Hospital and assistant professor of pediatrics at Harvard Medical School in Boston, Massachusetts.

CRISPR/Cas9 also could help maximize efficacy and minimize unwanted toxicity of cellular immunotherapy in acute myeloid leukemia (AML). Researchers have proposed that, by knocking out the AML antigen CD33 in normal hematopoietic stem cells (HSCs), CD33-directed immunotherapy can be used against AML without disrupting normal myeloid function.16

Correcting Genetic Defects

While cellular immunotherapy is the first application of CRISPR/Cas9 being evaluated in the clinic, Dr. Bauer pointed out that “not far behind may be applications for CRISPR in HSCs, where a variety of nonmalignant blood disorders could be ameliorated by permanent genetic modification. In addition, genome editing that may target non-hematopoietic cell types, such as hepatocytes that are more readily accessible to in vivo delivery, could address disorders of plasma factors, like hemophilia.”

Initially, therapies would be ex vivo, meaning HSCs would be removed from the patient, then edited with CRISPR/Cas9 and transplanted back into the patient. The edited stem cells would hopefully engraft into the bone marrow and overcome disease by producing healthy blood or immune cells. The advantage of this method is that the edited cells can be screened for correct repair and then enriched for transplantation.

However, extended manipulation and culture ex vivo may negatively impact cellular phenotype and engraftability. In vivo gene editing would overcome such limitations, but there are still technical challenges to resolve before in vivo therapies can be translated to the clinic.4,5

Already, proof-of-principle studies in human cells and animal models have shown that CRISPR/Cas9 can effectively correct hematologic genetic defects. Scientists use CRISPR/Cas9 with a DNA template containing a wild-type gene sequence to take advantage of HDR for precise gene correction. Disorders affecting single genes, including β-hemoglobinopathies (e.g., SCD and β-thalassemia) and immunodeficiencies (e.g., severe combined immunodeficiency), are most amenable to study.4,5,9,17

Diving Into Disease Development

“Perhaps even more transformational than therapeutic genome editing will be the use of CRISPR to deeply investigate the genetic underpinnings and modifiers of the gamut of blood disorders,” Dr. Bauer proposed. CRISPR technologies allow high-throughput and high-resolution modifications of sequences throughout the genome to determine the function and structure of genes and non-genic elements, he explained. “CRISPR will likely improve mechanistic understanding of pathophysiology and yield better disease models that, in turn, promise to accelerate the development of rationally designed pharmacotherapies.”

Dr. Goodell, who uses CRISPR/Cas9 to investigate leukemia, agrees. “We can use CRISPR to rapidly mutate different residues of a gene to determine whether particular domains are important or not for pathogenesis,” she said. “This has nearly immediate potential to identify druggable targets.” She added that CRISPR has been used in mice to reproduce leukemias, allowing researchers to test the importance of mutations in different genes, some of which are not yet known. “The technology has the potential to touch all aspects of leukemia (and more broadly, hematology) research, accelerating it significantly.”

“Many of us believe
engineered cell–
and stem cell–based
therapies are … the
next generation of
curative therapeutics.”

—Matthew Porteus, MD, PhD

Limitations of CRISPR/Cas9

The potential for CRISPR/Cas9 to reshape hematology is great, but “CRISPR alone is not enough,” Dr. Porteus noted. “It needs to be combined with multiple other tools and processes to have the impact that people are excited about. Sometimes this aspect gets overlooked in descriptions of how the Cas9/sgRNA system has changed what we are able to do.”

For example, Dr. Bauer added, “The delivery of genome-editing reagents (through viral transduction, physical means of delivery, etc.) may carry intrinsic risks and challenges that are not directly associated with CRISPR editing per se.”

Efficiency and Off-Target Effects

The limitations of genome editing “largely relate to potential off-target effects and efficiency of a particular editing procedure,” Dr. Bauer said. With these technologies, he asked, “can one edit sufficient numbers of cells to make a difference in phenotype?”

If the sgRNA recognizes a sequence outside the target gene, off-target editing or binding could cause unwanted genetic mutations (genotoxicity) and even malignant transformation. Furthermore, natural genetic variation in any given patient may disrupt the target site or introduce new off-target sites, thus affecting a therapy’s safety or efficacy. A paper published in Nature Medicine recommends screening for such genetic variation before giving patients CRISPR-based therapeutics.17

However, Dr. Bauer noted, “these concerns seem manageable with existing versions of the technology and likely will be further moderated with anticipated refinements. Like any novel therapeutic intervention, a risk-benefit calculation considering alternative treatment options will be critical to evaluating the appropriate use of CRISPR in the clinic.”


As with any revolutionary therapy, cost is a limiting factor for therapeutic CRISPR/Cas9 genome editing. “Cost and cost-effectiveness are important considerations as we consider how to distribute these sorts of novel therapies broadly to meet unmet clinical needs,” said Dr. Bauer. Alternative strategies for managing affordability of gene therapies include outcomes-based agreements in which payment levels are linked to the real-world outcomes achieved by patients, and payment amortization in which smaller payments are made over time.2

The Future of CRISPR Technology

The CRISPR/Cas9 toolkit is still expanding as new reagents are developed. “Areas of great interest are methods to maximize on-target editing efficiency, control genome-editing outcomes (such as via templated or non-templated repair pathways and  via base editing in the absence of double-strand breaks), minimize genotoxicity, and improve cellular delivery both ex vivo and in vivo,” Dr. Bauer said.

In vivo delivery of genome editing reagents would greatly simplify complex ex vivo cellular manufacturing approaches,” he added. “Ideally, CRISPR-based therapies could be re-dosable and titrated to clinical effect.”

Dr. Porteus described in vivo editing for hematologic disorders as “something of a holy grail,” and noted that one would have to find a highly effective delivery vector for the process to work. Dr. Bauer agreed that “understanding immune responses to CRISPR would be of great importance for allowing any in vivo delivery.”

“While there is tremendous excitement and potential, like any new technology, there will likely  be setbacks and the path to widespread benefits will be slower than everyone would want,” said Dr. Porteus. “That said, many of us believe that engineered cell– and stem cell–based therapies are going to be the next generation of curative therapeutics. There are still many scientific, translational, clinical, and commercialization questions that do not have answers and it will only be over the next decade that we begin to get those answers.”—By Amy Dear, PhD


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