CRISPR made even more precise, used to correct mutations linked to disease risk

Researchers have developed a modified version of CRISPR-Cas9 that can edit single letters of DNA without breaking the double helix, according to a paper in Nature. The authors say their method is more efficient and has fewer off-target effects than previous methods, demonstrating its potential by correcting single letter mutations linked to an increased risk of late-onset Alzheimer’s and cancer in lab grown cells.

Dr. Kris Saha, Assistant Professor of Biomedical Engineering, University of Wisconsin-Madison (webpage)

Expertise: gene editing, synthetic biology, human stem cell engineering, disease modeling, biomaterials.

“This work is a smart and elegant example of protein engineering. The work builds on new structural biology studies of the CRISPR system and a recent explosion of different protein fusions to Cas9, which range from fluorescent proteins to epigenomic writers. CRISPR-Cas9 is used essentially as a shuttle to bring in precise genome editing machinery to a specific sequence in the human genome. The approach sidesteps traditional error-prone DNA repair pathways that have been the focus of much of the work in the field for four years now. Instead, the team was able to fuse and exploit an alternative set of enzymes that cells in our body naturally use to fight viruses.”

“All of the work was performed within test tubes or cultured cells, so more studies will be needed to see whether the approach could be useful inside the body. Within the clinic, there is likely to be bottlenecks in delivering the fusion protein to complex tissues, like the brain. The system may be particularly useful in cells where components of the DNA repair machinery are limiting. Not all mutations can be edited with the strategy: only “Cs” (cytidines) in the genome are edited by the new base editor proteins. More protein engineering is needed to address “A” and “T” mutations with this system.”

“Even with cultured cells, the impacts of enhanced editing are likely to be significant. Gene editing has already been used to identify drug targets within cancer cells and help map out mechanisms of toxicity by various pathogens. I am particularly excited to see how the precise edits advanced by this technology can be used within human cells to refine and evolve genetic parts that have been developed by synthetic biologists. Our control of synthetic and developmental circuits is still quite rudimentary within human cells, so this work may eventually be an important tool for many engineering applications.”

“The ‘base editor’ proteins are shiny, new scalpels to perform genome surgery, which are specialized and sharp enough to precisely cut out specific sequences in hard-to-reach cells. They will be important tools for engineers and scientists, and perhaps eventually clinicians, to generate designer genomes within a wide variety of cells, including human cells.”


Dr. Perry Hackett, Professor, Center for Genome Engineering, University of Minnesota (webpage):

Expertise: Gene-editing techniques applied to both humans and animals.

“The paper out of David Liu’s lab by Kormor et al. is extremely important. It demonstrates that a defective Cas9 nuclease can be used, in collaboration with RNA guide sequences, to edit specific bases in genomic DNA. Classical gene editing techniques such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9, introduce breaks in the DNA double helix and then replace defective sequences by using a template sequence that has the desired changes in it. However, in this paper Komor et al. chemically transform specific cytosines (the ‘C’ in the chemical bases A, T, G, C that make up DNA) in the target sequence so that it can be replaced with an adenine(A) without introducing a break in the DNA. Alternatively, by editing a C on the other strand of DNA, a G can be converted to an A.

“One problem that the authors recognize is that if you have two C’s next to each other in the genome and you only want to change one of them, the reliability will be low since the enzyme may target one or the other C. In every gene-editing technique however, the nature of the target sequence greatly influences the efficiency. Relatively speaking, the new approach designed by Komor et al only modestly increases the efficiency of gene editing over CRISPR-Cas9. However it does demonstrate fewer off target effects and greater specificity, which is a significant advance.

“All of us are amazed at how fast this technology is developing and we keep coming up with new applications all the time. However, all of these techniques will take time for validation so it is not clear when these technologies will be used in therapeutic applications.”


Dr. Mathew Blurton-Jones, Assistant Professor, Neurobiology and Behavior, School of Biological Sciences, University of California Irvine (webpage):

Expertise: Parkinson’s Disease, Alzheimer’s Disease, stem cell therapies

“The data presented by the authors is quite convincing and supports their approach towards significantly improving the efficiency and specificity over current CRISPR-Cas9 gene editing approaches.

“Whether this approach could actually be adapted to a direct clinical application is unclear. It would be very difficult to do such reprogramming in vivo, although some studies are demonstrating some potential for this. Rather, the more important use of this technology in the foreseeable future will be to provide a powerful tool to study the influence of specific genes on the function of cells and their role in disease.

“As an example, the authors use their approach to edit the Alzheimer’s Disease (AD) risk gene Apolipoprotien E (ApoE) in a mouse cell line that harbors a human version of this gene. It is well established that the ‘E4’ version of this ApoE gene increases the risk of developing AD. The authors therefore used their programmable editing technique to modify the genome of a cell to convert its ApoE gene from this ‘E4’ version to the more common ‘E3’ version. In the current paper, the authors do not examine how this alters the function of these cells. However this is precisely how this technology can be used to improve our understanding of diseases such as AD.

“For example, by using this kind of approach to edit ApoE in human astrocytes (a type of cell in the brain), researchers could further advance our understanding of how ApoE genetic variants change the function of these cells and contribute to the development of AD.”


Dr. Mark Osborn, Assistant Professor, Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota (webpage):

Expertise: Gene Therapy, Genome Editing, Cellular Therapy

“The study by Komor et al. describe a ‘base-editing’ approach for genome modification using a version of Cas9 that is fused to an enzyme that results in a C→T (or G→A) modification at the DNA target site. Using this system, the authors show that the APOE4 gene can be modified in cell cultures to a genetic variant that is associated with a lower risk for Alzheimer’s disease. This represents a novel application of the CRISPR-Cas9 platform and a new tool in genome engineering. This platform shows enhanced specificity with minimal non-specific insertions and deletions at the target site. This fact, coupled with the ability to modify bases without need of a repair template sequence (as is needed by programmable nucleases) represents an important advance that may speed therapeutic application. DNA-based donor templates externally introduced into the cell for precision alterations can also integrate at random or semi-random locations and by minimizing the components needed for base modification the safety and efficacy can be maximized.”



Declared interests (see GENeS register of interests policy):

Dr. Perry Hackett: Dr. Hackett is Chief Scientific Officer at Recombinetics, a company that uses gene-editing techniques in animals but not humans.

No further interests declared.



Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage‘ by Alexis C. Komor et. al., published in Nature on Wednesday, 20 April 2016.

Please feel free to leave your comments below, but be aware that by doing so you agree to our Terms & Conditions.