A New Tool for Gene Editing In Stem Cells Can Drive Changes in Cell Fate Without Causing Mutations


Recently, a new tool is now available to control gene expression in order to understand gene function and manipulate cell fate. This new tool is called CRIPSR/Cas9, which is a gene-editing tool that employs a genetic system that naturally occurs in bacteria, who use it as a protection against viruses. CRISPR/Cas9 allows scientists to precisely add, remove or replace specific sequences of DNA. It is the most efficient, inexpensive and easiest gene editing tool available to date.

Several laboratories have tried to use CRISPR/Cas9 to activate genes in cells, but such an effort has not always succeeded. However a research team at Hokkaido University’s Institute of Genetic Medicine has developed a powerful new method that uses CRISPR/Cas9 to do exactly that.

In cells, genes have an expression switch called “promoters.” Genes are switched off, or silenced, when their promoters are methylated, which means that islands of C-G bases have a methyl group (a –CH3 group) attached to the cytosine base. The Hokkaido University team wanted to turn an inactivated gene on. The ingeniously combined a DNA repair mechanism, called MMEJ (microhomology-mediated end-joining), with CRISPR/Cas9 to do this. They excised a methylated promoter using CRISPR/Cas9 and then used MMEJ to insert an unmethylated promoter. Thus, they replaced the off-switch signal with an on-switch signal.

DNA Methylation

The gene that was activated was the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in order to test the efficiency of this technology in cultured cells. Within five days, they found evidence that the genes were robustly expressed. When they activated the OLIG2 gene in cultured human stem cells, the cells differentiated to neurons in seven days with high-efficiency.

Toru Kondo and his colleagues also discovered that their editing tool could be used to activate other silenced promoters. They also found that their system didn’t cause unwanted mutations in other non-target genes in the cells. According to Kondo, this gene editing tool has wide potential to manipulate gene expression, create genetic circuits, or to engineer cell fates.

See Shota Katayama et al., “A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation,” Angewandte Chemie International Edition, 2016; 55(22): 6452 DOI: 10.1002/anie.201601708.

Accelerated Reprogramming and Gene Editing Protocol Can Make Fixed Cells Much Faster


Sara Howden and her colleagues at the Morgridge Institute for Research and the Murdoch Children’s Research Institute in Australia have devised a protocol that can significantly decrease the time involved in reprogramming mature adult cells while genetically repairing them at the same time. Such an advance is essential for making future therapies possible.

Howden and others demonstrated that genetically repaired cells can be derived from patient skin cells in as little as two weeks. This is much shorter than the multistep approaches that take more than three months.

How were they able to shorten the time necessary to do this? They combined two integral steps in the procedure. Adult cells were reprogrammed to an embryonic stem cell-like state in order to be differentiated into the cells that we want. Secondly, the cells must undergo gene editing in order to correct the disease-causing mutation.

By in this new protocol developed by Howden and her colleagues, they combined the reprogramming and gene editing steps.

To test their new protocol, Howden and her team used cells isolated from a patient with an inherited retinal degeneration disorder, and an infant with severe immunodeficiency. In both cases, the team not only derived induced pluripotent stem cell lines from the adult cells of these patients, but they were also able to repair the genetic lesion that causes the genetic disease.

This protocol might advance transplant medicine by making gene-correction therapies available to patients in a much timelier fashion and at lower cost.

Presently, making induced pluripotent stem cell lines from a patient’s cells, genetically repairing those cells, expanding them, differentiating them, and then isolating the right cells from transplantation, while checking the cells all along the way and properly characterizing them for safety reasons would take too long and cost too much.

With this new approach, however, Howden and others used the CRISPR/Cas9 technology to edit the damaged genes while reprogramming the cells, greatly reducing the time required to make the cells for transplantation.

Faster reprogramming also decreases the amount of time the cells remain in culture, which minimizes the risks of gene instability or epigenetic changes that can sometimes occur when culturing cells outside the human body.

Howden’s next goal is to adapt her protocol to work with blood cells so that blood samples rather than skin biopsies can be used to secure the cells for reprogramming/gene editing procedure. Blood cells also do not require the expansion that skin cells require, which would even further shorten the time needed to make the desired cell types.

The accelerated pace of the reprogramming procedure could make a genuine difference in those cases where medical interventions are required in as little time as possible. For example, children born with severe combined immunodeficiency usually die within the first few years of life from massive infections.

Howden cautioned, however, that she and her team must first derive a long-term source of blood cells from pluripotent stem cells before such treatments are viable and demonstrate the safety of such treatments as well.

See Stem Cell Reports, 2015: DOI: 10.1016/j.stemcr.2015.10.009.

Researchers Grow Retinal Ganglion Cells in the Laboratory


Researchers from laboratory of Donald Zack at The Johns Hopkins University in Baltimore, Maryland have used genome editing methods to efficiently differentiate human pluripotent stem cells into retinal ganglion cells. Retinal ganglion cells are found in the retina that and helps transmit visual signals from the eye to the brain. Abnormalities or death of ganglion cells can cause vision loss, and conditions such as glaucoma and multiple sclerosis can wreak havoc on ganglion cells.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” said Zack, who is the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

Published in the journal Scientific Reports, Zack and his team genetically modified a line of human embryonic stem cells so that they would fluoresce once they differentiated into retinal ganglion cells. Then they used these cells to develop new differentiation methods and characterize the resulting cells.

To genetically modify their cells, Zack and others used the CRISPR-Cas9 system. CRISPR stands for “clustered regularly interspaced short palindromic repeats” and these are short segments DNA, which are found in bacteria, contain short repeated sequences. Following each repeated sequence is a short spacer that usually comes from previous exposures to a bacterial virus or plasmid. Bacteria use the CRISPR/Cas system as a kind of immune system that prevents cells from being invaded by foreign DNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes.

When bacteria are invaded by a virus, the particular Cas nucleases capture the viral DNA, cut it and insert it into the CRISPR array. When the bacterial cell is infected by a virus, an RNA is transcribed from the CRISPR array called the crRNA. This crRNA then hybridizes with the invading DNA or RNA and the double-stranded RNA or DNA/RNA hybrid is degraded by Cas proteins.

The CRISPR/Cas system is a useful laboratory tool for gene editing or adding, disrupting or changing the sequences of particular genes. If Cas9 and the appropriate crRNA are delivered into cells, you can cut a genome almost anywhere. CRISPR has a huge number of potential applications.

Zack and his group used the CRISPR/Cas system to insert a fluorescent protein gene into the DNA of their stem cells line. This red fluorescent protein would be expressed if a gene called BRN3B (POU4F2) was also expressed. BRN3B is expressed by mature retinal ganglion cells. Therefore, once these cells differentiated into retinal ganglion cells, they would glow red when viewed with a fluorescence microscope.

After differentiating their cells, Zack and his coworkers used a technique called fluorescence-activated cell sorting to isolate fully differentiated cells from other cells. The pure cell culture contained cells that displayed the biological and physical properties observed in retinal ganglion cells produced naturally, according to Zack.

As an added bonus, Valentin Sluch, a former graduate student in Zack’s laboratory, and her colleagues discovered that soaking the pluripotent stem cells in a chemical called “forskolin” at the commencement of the differentiation protocol significantly improved the efficiency of differentiation. Forskolin is a labdane diterpene found in the roots of the Indian Coleus plant (Coleus forskohlii), which belongs to the mint family.  It is used by some people as a weight loss supplement by some people.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” said Sluch, who is now a postdoctoral scholar working at Novartis. Sluch continued, “I was very excited when it first worked. I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for Multiple Sclerosis, Zack is collaborating with Dr. Peter Calabresi, professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

Correcting Mutations Associated with a Blood Disorder


The protein hemoglobin carries oxygen from our lungs to our tissues. Mutations in the genes that encode the protein chains that form hemoglobin can cause inherited blood disorders like sickle-cell anemia, or the so-called Thalassemias. Thalassemias come from the Greek word from sea (θάλασσα or thalassa), because these blood disorders are found in Mediterranean populations. Thalassemias are found in these populations because they convey some resistance to malaria, which was endemic to that area. People with thalassemias tend to have fatigue, weakness, a pale appearance, yellow discoloration of skin (jaundice), facial bone deformities, slow growth, abdominal swelling, or dark urine, although some people have no symptoms.

Now this common genetic blood disorder has been genetically corrected in cultured induced pluripotent stem cells by using cutting-edge genome-editing techniques.

β-Thalassaemia shows reduced levels of hemoglobin, and these reduced levels are due to mutations in the gene that encodes the β-globin protein. Hemoglobin consists of four protein chains, two of which are alpha-globin proteins, and the other two of which are beta-globin proteins. Mutations in the beta-globin gene reduces the levels of functional beta-globin protein and this reduces the levels of functional hemoglobin.

Yuet Kan and his colleagues at the University of California, San Francisco, made induced pluripotent stem cells from skin fibroblasts from a person who suffered from β-thalassemia. Kan and his colleagues then used the CRISPR–Cas9 gene-editing technique to correct the mutation responsible for β-thalassemia. The CRISPR–Cas9 gene-editing technique allows for precise and accurate correction of the mutation without affecting other genes.

After the genetic editing, the iPSCs were differentiated into the precursors of red blood cells in culture and demonstrated that the modified cells showed higher expression of hemoglobin than unmodified cells.

Hopefully transplantation of such corrected cells back into the original patient could one day provide a cure for β-thalassaemia, according to the authors.