一道本不卡免费高清A team of scientists at UC San Francisco and the National Institutes of Health have achieved another CRISPR first, one which may fundamentally alter the way scientists study brain diseases.
In a paper published Aug. 15 in the journal , the researchers describe a technique that uses a special version of CRISPR developed at UCSF一道本不卡免费高清 to systematically alter the activity of genes in human neurons generated from stem cells, the first successful merger of stem cell-derived cell types and CRISPR screening technologies.
一道本不卡免费高清Though mutations and other genetic variants are known to be associated with an increased risk for many neurological diseases, technological bottlenecks have thwarted the efforts of scientists working to understand exactly how these genes cause disease.
“Prior to this study, there were significant limitations that restricted what scientists could do with human neurons in the lab,” said , PhD, associate professor in UCSF’s , a CZ Biohub Investigator, and co-senior author of the new study.. “It was possible to get neurons donated by patients who had undergone procedures that involve removing brain tissue to treat epilepsy or brain cancer. But these samples can only survive for a few days. You can’t perform experiments to probe gene function on short-lived neurons.”
一道本不卡免费高清Instead, scientists have generally relied on animal models of brain disease, which can fail to capture many nuances of human neurobiology.
A breakthrough came in 2006 when , MD, PhD, of Kyoto University and the UCSF-affiliated , discovered a way to rewind the developmental clock and turn adult cells into stem cells that could, with some coaxing, be transformed into any type of cell found in the body – including neurons. These “induced pluripotent stem cells” (iPSCs) made human brain cells widely available for lab research.
一道本不卡免费高清When the CRISPR gene-editing system arrived six years later, scientists thought they finally had all the tools they would need to manipulate genes in human neurons and determine how they contribute to neurological disease.
一道本不卡免费高清But scientists quickly discovered that the DNA-cutting machinery of the CRISPR system, an enzyme known as Cas9, didn’t mix well with iPSCs. “Stem cells have a very active DNA damage response. When Cas9 produces even just one or two DNA cuts, it can lead to toxicity that causes the cells to die,” Kampmann said.
一道本不卡免费高清So Kampmann decided to tackle the toxicity problem. As a postdoc in the lab of UCSF professor , PhD, Kampmann co-invented a tool known as CRISPRi (for “interference”), a modified form of CRISPR technology in which the Cas9 enzyme has been deactivated. When CRISPRi finds the gene it’s seeking, it suppresses its activity without making any cuts. As a result, unlike standard CRISPR-Cas9, Kampmann predicted, CRISPRi shouldn't be toxic to iPSCs or stem cell–derived neurons.
In the new paper, Kampmann and his collaborators describe how they adapted CRISPRi for use in human iPSCs and iPSC-derived neurons, and found that it could target and interfere with genes without killing the cell – a feat that had long eluded scientists.