CRISPR-LIGHT- Novel Technology in Brain Organoids

CRISPR-LIGHT- Novel Technology in Brain Organoids

Release Date: 29-Nov-2020



Scientists have developed a new screening technique which uses organoids to test the effect of gene mutations on brain. To recognize the gene which control the cell growth, scientists have developed a variety of disruptive mutations and then measured the effect of gene on cell proliferation. CRISPR technology was used to  introduce different ‘guide RNA’ snippets into a pool of cells. The guide RNAs get integrated into the cells’ genomes and escort an enzyme, such as Cas9, to a specific spot to cut the DNA. After a period of time, researchers sequence DNA from the cells to determine the abundance of each guide RNA and infer which ones altered genes involved in cell proliferation.

 

The loss of function requires all the cells to divide at the similar rates. However, the cells in the brain organoids fails to do so. To overcome this, scientists have developed DNA barcodes to tag individual organoids and each cell within each organoid. This barcoding enables them to track the effects of any guide RNA across hundreds of organoids, providing a clearer snapshot of its overall effects. The novel technique is called CRISPR-LIGHT.

 

For this, the researchers first engineer human stem cells to produce a version of the DNA-cleaving enzyme Cas9. They then use viruses to introduce an array of guide RNAs into the stem cells. The guide RNAs are designed to introduce damaging mutations into genes of interest. The viruses also ferry a unique DNA tag, or barcode, into each cell’s genome, enabling the researchers to keep tabs on which cells contain which guide RNAs.

 

Further, the separation of the stem cells is done to grow them into the organoid. After 40days, the DNA is extracted from the cells and then add a second barcode which allows to count the number of organoid a cell has. The study analyzed 172 genes which are linked with microcephaly, or small head size andmdash; for their ability to control organoid growth. Overall, mutations in 25 genes significantly decreased cell proliferation, suggesting they play a role in the condition, the researchers report. 

 

Most of identified genes have role in biological process involved with microcephaly, but some are involved in lesser-known pathways, such as regulating protein secretions from the endoplasmic reticulum, the cell’s protein factory. Brain organoids lacking a functional copy of a gene involved in this pathway are smaller in size and have reduced structural protein levels compared with organoids that have functional copies of the gene. The researchers hope that CRISPR-LICHT could be used to screen other gene functions, and could help scientists decipher how autism-linked genes affect brain organoids.

 

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