Dana Carroll, PhD, is a distinguished professor in the department of biochemistry at the University of Utah School of Medicine in Salt Lake City. A member of the Nuclear Control of Cell Growth and Differentiation Program at the university’s Huntsman Cancer Institute, he pioneered gene editing with zinc-finger nucleases, and is a leading authority on other genome-editing tools, including the Streptococcus bacteria-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) platform. Cas9 is a DNA endonuclease enzyme; CRISPR-Cas9 is a bacterial adaptive immune system that identifies and disables detected viral genes. Dr Carroll is collaborating with researchers at the University of California, Berkeley, and the Oakland Children’s Hospital Research Institute to develop a CRISPR-based treatment for the mutation underlying sickle-cell disease.

In this question-and-answer session, Cancer Therapy Advisor asked Dr Carroll about CRISPR-Cas9, the state of the science of genome editing, and its implications for oncology.

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Cancer Therapy Advisor (CTA): Oncologists are hearing a lot about genome editing: zinc-finger nucleases, Transcription Activator-Like Effector Nucleases (TALENs), and, of course CRISPR-Cas9. What are the advantages and disadvantages of the different genome-editing nuclease platforms?  

Dr Carroll: This question deserves a long answer, but I’ll try to give a brief 1 without doing violence to the complexities. CRISPR-Cas9 is the easiest platform to use. To target a new genomic sequence, one needs only to produce a new guide RNA based on the simple principle of Watson-Crick base pairing. Multiple targets can be addressed simultaneously or sequentially by providing Cas9 along with several or many individual guide RNAs.

Both ZFNs and TALENs require the design and production of 2 new proteins for each target being attacked. The TALEN and CRISPR-Cas9 platforms have shown very high success rates, i.e., more than half of all new designs work well.

This is not true of ZFNs, at least in the public sphere, because the DNA-recognition modules (zinc fingers) have variable and unpredictable properties. Scientists at Sangamo Biosciences have developed databases and methods that allow them to generate new designs with greater success. Nonetheless, ZFNs have 2 properties that make them attractive: they are rather small proteins, so their coding sequences fit comfortably in viral vectors with limited capacity, and they have been in clinical trials for several years already. The experience with them is also encouraging.

CTA: Many people refer to CRISPR as a single genome-editing platform. Are there different versions of CRISPR in use? What can you tell us about those?  

Dr Carroll: There are lots of variations on the original Streptococcus pyogenes Cas9 system. Other Cas9 proteins have been used, such as from Staphylococcus aureus, and there are more distantly-related proteins that have similar properties, most notably Cpf1. Variants of Cas9 have been engineered to recognize alternative classes of sequences or to have improved specificity.

In addition, there are modified versions of Cas9 that have no nuclease activity, and are designed to regulate gene expression by binding to specific sequences in the genome.