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Pregnancy Timeline by SemestersFetal liver is producing blood cellsHead may position into pelvisBrain convolutions beginFull TermWhite fat begins to be madeWhite fat begins to be madeHead may position into pelvisImmune system beginningImmune system beginningPeriod of rapid brain growthBrain convolutions beginLungs begin to produce surfactantSensory brain waves begin to activateSensory brain waves begin to activateInner Ear Bones HardenBone marrow starts making blood cellsBone marrow starts making blood cellsBrown fat surrounds lymphatic systemFetal sexual organs visibleFinger and toe prints appearFinger and toe prints appearHeartbeat can be detectedHeartbeat can be detectedBasic Brain Structure in PlaceThe Appearance of SomitesFirst Detectable Brain WavesA Four Chambered HeartBeginning Cerebral HemispheresFemale Reproductive SystemEnd of Embryonic PeriodEnd of Embryonic PeriodFirst Thin Layer of Skin AppearsThird TrimesterSecond TrimesterFirst TrimesterFertilizationDevelopmental Timeline
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Home | Pregnancy Timeline | News Alerts |News Archive Aug 29, 2013

 

Cas9

Whitehead Institute researchers created the enzyme used in CRISPR-on
by fusing the Cas9 protein to a VP160 domain containing 10 tandem copies
of VP16 motifs. The VP160 domain acts as a transcriptional activation domain.

Image: Courtesy of Cell Research.





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New approach to gene regulation can activate multiple genes simultaneously

By creating a powerful new gene regulation system—CRISPR-on— Whitehead Institute researchers have the ability to increase the expression of multiple genes simultaneously and precisely, as well as be able to manipulate each gene's expression level. The system is effective in both mouse and human cells.

"CRISPR-on is a tool that will be very useful for studying many biological processes, particularly for studying gene functions and gene networks," says Whitehead Founding Member Rudolf Jaenisch. "In contrast to RNA interference, which is commonly used to inactivate gene activity, the CRISPR-on system allows activation of cellular genes. The technology substantially expands our ability to change gene expression in cultured cells and animals."

The system, called CRISPR-on, is a modified version of CRISPR/Cas (for "clustered regularly interspaced short palindromic repeat/CRISPR associated"), which taps into a bacterial defense system against viral intruders. CRISPR/Cas relies on an enzyme, Cas9, which cuts DNA at locations specified by single guide RNAs (sgRNAs).


For CRISPR-on, the Whitehead team modified the Cas9 enzyme—by eliminating its ability to cleave DNA—and adding a transcription activation domain.

The resulting enzyme can increase gene expression without permanently changing the DNA.


The new system is described this week in the journal Cell Research.

CRISPR-on's ability to activate only the desired genes at varying levels could be used to help scientists improve our understanding of transcription network underlying a variety of diseases and potentially find new ways to treat them.


"Many diseases, especially complex diseases, involve multiple genes. This system could be used therapeutically to target and activate multiple genes together and rescue these disease phenotypes. Or we could use it to study the gene networks in diseases and get a better understanding of how those diseases work."

Albert Cheng, graduate student in the Jaenisch lab, co-author of the Cell Research paper.


So far, the researchers have used CRISPR-on to activate up to three native genes concurrently in human cells.

"I think we need to do more work to see if there are any limitations to the number of genes CRISPR-on can activate at a time," says Haoyi Wang, a co-author and postdoctoral researcher in the Jaenisch lab. "We'd like to see if we can get data on activating 10 or more genes, to see if there is an upper limit to what this system can do."

Abstract
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.

10.1038/cr.2013.122

This work is supported by the Croucher Foundation and National Institutes of Health (NIH) grants HD 045022 and R37CA084198.

Rudolf Jaenisch's primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

Full Citation:

"RNA-guided multiplexed endogenous gene activation"

Cell Research, online August 27, 2013.

Original press release: http://www.eurekalert.org/pub_releases/2013-08/wifb-nat082613.php