Some genetic sequencing misses out large parts of the genome
Before this study the team at Broad Institute has been working on genetic libraries of data that could be used to look at the results of removal of one or more genetic sequences. Finally they were ready with libraries that could selectively turn on or off any gene in the mice brain. They used a particular short hairpin RNA (shRNA) genetic library to look at messenger RNA which carries vital information for protein synthesis. They used CRISPR to delete or edit the genetic sequences and used viral carriers to carry within the cell the necessary altered genetic sequences.
The four or five shRNAs or CRISPR segments targeted each of the 22,000 mice brain genes using approximately 80,000 to 100,000 viruses to alter each of the brain cells within the mice. The viruses carrying the segments were taken in high concentration and injected within the brain in the striatum region and at least a quarter of all the brain cells received at least one of the shRNA or CRISPR elements. The striatum region was more of a target because it deals with the motor system of the body and helps in movement as well as cognitive functions and emotions. This area is not only affected by Huntington’s disease but also autism, Parkinsonism and drug addiction.
The mice were injected with viral carriers for seven months continuously after which the genetic makeup of the neurons of the stratum was checked. The neurons that needed the knocked out genes for survival would have died by the end of the study, explained the researchers. On the other hand if non-essential genes were knocked out, the neurons would be alive.
Results showed that several genes were important for the neurons to survive. This also yielded several genes vital for survival of the neurons but unknown in previous studies. Heiman said that they found certain genes that were not known earlier to be important. She added, “What we interpret this to mean is that neurons in the mammalian brain are much more metabolically active and have a much higher dependency on these processes than for example, a neuron in C. Elegans.”
They noted results in mice models that produced mutated form of the huntingtin protein. The screening of the normal mice was compared with those with Huntington's disease. If these mice contained less amounts of any shRNA or CRISPR elements, these could be deduced to be important targets that resisted the toxic effects of the huntingtin protein explained Heiman. The team found that drugs targeting the Nme1 gene could be one such target.
Heiman concluded, “This is very exciting to us because it's theoretically a druggable compound. If we can increase its activity with a small molecule, perhaps we can replicate the effect of genetic overexpression.” This study was supported by the National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke (NINDS) and others. Journal reference:
Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity Wertz, Mary H. et al. Neuro, https://www.cell.com/neuron/fulltext/S0896-6273(20)30004-0
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