To use Cas9 to edit genes

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biokit
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To use Cas9 to edit genes

Post by biokit » Fri Sep 02, 2016 8:30 am

Cas9, a naturally occurring protein in the immune system of certain bacteria, acts like a pair of molecular scissors to precisely cut or edit specific sections of DNA. The CRISPR-Cas9 system has been in the limelight mainly as a revolutionary genome engineering tool used to modify specific gene sequences within the vast sea of an organism's DNA. However, recently, scientists have also begun to use CRISPR-Cas9 variants as gene regulation tools to reversibly turn genes on or off at whim.

Before, genome engineering and gene regulation are initiated with a common step: the Cas9 protein is recruited to targeted genes by the so-called matching sequences of "guide RNA" that help Cas9 latch on to specific sequences of DNA in a given genome. But now, they require different variants of the Cas9 protein; while the former task hinges on Cas9's innate DNA-cleaving activity, the latter has been achieved by engineered Cas9 variants that have had their DNA-cleaving "fangs" removed, but still retain their ability to latch onto a specific genomic target. The latter Cas9 variants are commonly fused with proteins that regulate gene expression.

Now, both tasks can be achieved using one type of Cas9 using a new approach, allowing scientists to increase the complexity of gene editing functions and their overall control of genes. This approach was developed by researchers led by George Church, Ph.D., of Harvard and Ron Weiss, Ph.D., of the Massachusetts Institute of Technology. The method opens up unexpected possibilities for understanding diseases and drug mechanisms.

The multi-institutional team has introduced the clever new kit that allows the innate Cas9 protein from Streptococcus pyogenes to cleave certain genes while simultaneously regulating the expression of others through engineering the guide RNA. In their study, they found that the length of the guide RNA sequence plays a critical role in determining whether or not Cas9 will solely bind to DNA or if it will excise it as well. So they systematically test why it was that truncating guides too much caused Cas9 to no longer cut the intended genomic site.

The Wyss and MIT team confirmed in human cells that shorter guide RNAs indeed no longer allowed Cas9 to cut a targeted gene. To their surprise, however, the shorter guide RNAs did not prevent Cas9 from efficiently binding to that target, opening up the possibility for scientists to attach gene regulation proteins to Cas9 for delivery to specific genes.

This new functionality will improve ability to decipher the complex relationships between interdependent genes responsible for many diseases, and the findings can be used in large scale metabolic production of chemicals and fuels using genetically engineered bacteria. Hope that Cas9 can be a revolutionary tool allowing scientists to conquer new biomedical and industrial territory. The study's findings are reported in the September 7 issue of Nature Methods.

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