ASH Clinical News FINAL_ACN_3.14_FULL_ISSUE_DIGITAL | Page 104

Demystifying the Lab ASH Clinical News takes a look at the complex scientific techniques that hematologists/oncologists hear about every day, with practical information for the practicing clinician. 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 intro- ducing 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, ef- ficiency, and ease of use. 1 Gene therapy is predicted eventually to benefit millions of patients by enabling shorter treatment regimens with longer-lasting cura- tive 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 revolu- tion for science and for medicine,” Stuart Orkin, MD, associate chief of the division of hematol- ogy/oncology and chairman of the pediatric oncology department at Boston Children’s Hospital and David G. Nathan Professor of Pe- diatrics 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 tech- nology 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 hand.” —MARGARET GOODELL, PhD 102 ASH Clinical News 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 (see FIGURE 1 ). 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 disruption 4 • homology-driven repair (HDR), which aids in introducing novel DNA by exploiting donor DNA molecules that have homologous sequences surrounding the DNA break 5 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, including zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs; see FIGURE 2 ). 3 ZFNs ZFNs are engineered DNA-binding proteins that facilitate targeted genome editing by creat- ing double-strand breaks in DNA at specified locations. ZFNs consist of a FokI nuclease do- main 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 TALENs 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 CRISPR/Cas9 is the most recent gene editing tool to be added to the repertoire. It is adapted from a naturally occurring genome editing sys- tem 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 “remem- ber” 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 of- ten 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 cut- ting 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 pro- tein engineering for each new target. 4,5 “CRISPR is easy, efficient, and relatively inexpensive. This is such an accessible tech- nology 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 Regenera- tive Medicine Center in Houston, Texas. “The pace of experiments and the types of experi- ments 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.” 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 g ene therapies, which fall into the follow- ing 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 December 2017