Written by Navaneeth Mohan
A form of Autism—fragile X syndrome (FXS)—is caused by a repeating genetic sequence in the human brain. This prompted scientists to consider using CRISPR-Cas9, a molecular scissor, to prune the creeper at its roots. However, the conventional CRISPR-Cas9 is delivered via a virus which triggers the body’s immune system to eat up the CRISPR-Cas9 system. A team of researchers based in Texas and California found a golden alternative to the viral delivery system. Literally. CRISPR-Gold delivers the Cas9 and Cpf1 Ribonucleoproteins packaged in a gold nanoparticle. It is capable of editing mice brain cells with undetectable side-effects. Further, patient mice treated with CRISPR-Gold displayed up to 70% fewer symptoms of FXS.
Fragile-X syndrome (FXS) is a particularly debilitating disease due to the current lack of therapeutic options. It is the most common inherited form of intellectual disability under the umbrella of Autism Spectrum Disorders (ASD). It is caused by an abnormally high repetition (> 200 times) of the CGG nucleotide in the fragile-X mental retardation 1 (FMR1) gene .
To surgically excise the abnormal repetitions, scientists used a molecular scalpel: CRISPR, the gene editing mechanism that took the biomedical world by storm in 2012. A CRISPR associated protein 9 (Cas9) tagged with a homing beacon, in this case a single guide RNA (sgRNA), could specifically target and modify a nucleotide sequence. Furthermore, the ease of generating a sgRNA meant CRISPR was scalable. With a single intracranial injection, the genes pertaining to a host of neurological diseases could be edited, including mGluR5, which is expressed extensively in FXS patients [2,3].
However, traditional delivery methods for CRISPR-Cas9 and sgRNA cause genomic damage [4,5]. The viral packaging used to infiltrate the target’s DNA can continue churning out the DNA snipping CRISPR system. This prolonged expression of the CRISPR system can cause unintended edits to the DNA. Furthermore, the viral packaging causes the body’s immune system to attack the CRISPR-Cas9 system. A work-around would require a non-viral delivery mechanism.
A team of researchers from University of Texas Health Science Center at San Antonio, GenEdit Inc., Berkley, and University of California Berkley have manufactured this work-around . Entitled CRISPR-Gold, by substituting a gold nanoparticle for the virus, the team had previously delivered Cas9 , successfully, into mouse muscles . This encouraged them to investigate the efficacy of CRISPR-Gold as a delivery vector for gene editing in neuronal cells.
Preliminary biocompatibility tests on cultured neurons showed that, in comparison to a control culture, the culture treated with CRISPR-Gold Cas9 complex showed no significant changes in electro-physiological properties. Also, no significant differences were observed in the number of dead neurons and the cell morphology. Following this positive result, the team experimented on healthy mice.
Two genetically engineered mouse models were chosen. The Thy1-YFP mouse exhibits fluorescence only in the neurons . Knocking out the YFP gene defuses the fluorescence. The Ai9 mouse, on the other hand, has a fluorescent tdTomato gene silenced by a stop sequence . Deletion of the stop sequence re-activates the tdTomato gene’s fluorescence.
In both mouse models, injection of CRISPR-Gold complexes showed positive results. In Thy1-YFP mice, stereotaxic injection of CRISPR-Gold complex) targeting the 5’ region of the YFP gene caused a 25-34% decrease in YFP expression levels. A similar injection CRISPR-Gold complexes targeting the tdTomato stop sequence of Ai9 mice resulted in a 10-15% increase in tdTomato fluorescence.
It is well known that glial cells play a central role in maintaining neuronal function. Ai9 mice express fluorescence in glial cells as well. Hence, following the injection of CRISPR-Gold Cas9 or Cpf1 complexes, Ai9 mice brains were stained with glial markers such as glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA1). Among the fluorescent cells observed, ~90% had glial markers. This suggests that CRISPR-Gold complexes can edit glial cells, in addition to neurons.
Following the experiments on the healthy mice, the team targeted the mGluR5 gene of Fmr1 knockout mice (Fmr1 KO), a mouse model of FXS. Treatment withCRISPR-Gold complexes resulted in a 40-50% reduction in the mRNA and protein levels of mGluR5 gene. Even more promising was the phenotypic changes observed. In comparison to patient mice administered saline, the treatment group exhibited up to 70% fewer symptoms of OCD and anxiety such as repeated jumping or marble burying .
CRISPR-Gold delivery of Cas9 and Cpf1 clearly shows a promising venue for treatment of a wide host of neurological disorders. The complex is administered by intracranial injections, which can only target local regions. A global targeting method is necessary for a number of neurological disorders and is being researched. Despite this, Fmr1 KO mice showed significant phenotypic changes when treated such. Of particular interest was CRISPR-Gold’s ability to edit glial cells, which was previously challenging via viral delivery methods .
A question still open for debate regards the discharging of gold nanoparticles from the neurons. It is unclear whether accumulation of gold nanoparticles in the neurons leads to toxicity. Though a single dosage is harmless (2.84μg/kg), repeated injection of CRISPR-Gold might be unfeasible.
An even more worrisome side-effect of CRISPR’d cells is the off-target genome editing . Like any imperfect tracking system, the CRISPR’s sgRNA can bind to regions that only approximately complement the sgRNA. While researchers are currently investigating methods to reduce such off-target snips, the stringent regulations surrounding gene therapy keeps CRISPR based gene editing a distant dream .
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About the Author:
Navaneeth Mohan is an M.Sc. graduate of Western University in Canada. For his research, he investigated blood pressure fluctuations using tools from non-linear dynamics. Alongside his research, he is also an active science communicator. As a former member of Gradcast and freelance contributor at Western News, he has collaborated with researchers to produce podcasts and articles that deliver science to the populace.