CRISPR/Cas9 for engineering biology

Cas9 is a remarkable targeted DNA-cutting enzyme from Type II CRISPR systems. These elements provide bacteria with acquired immunity to bacteriophages and parasitic elements by storing fragments of DNA and cutting any sequences that exactly match the fragment. Cutting happens when RNA molecules made from the stored fragment are used to direct Cas9, an "RNA-guided nuclease", to cut matching double-strand DNAs.

By providing Cas9 with an appropriate guide RNA, we can direct it to bind and cut almost any sequence we want. If we also provide a repair template containing an edited version of the gene (see left), the cell can incorporate the edited version in place of the original.  This makes Cas9 a tremendously useful tool for genome editing. Scientists have successfully used it to alter the genomes of a wide variety of species, even making multiple edits at the same time.

Because Cas9 is such a versatile endonuclease, it might even be able to spread these alterations through populations.  We recently outlined how Cas9 could  might be used to build RNA-guided gene drives capable of spreading alterations through wild populations. The idea is simple: when editing a gene, include the cas9 gene and guide RNAs next to the edited version so that all of them are incorporated into the edited chromosome.  When passed on to future generations that also inherit a wild-type version of the gene, Cas9 will again cut the wild-type sequence, causing the edited gene, cas9, and guide RNAs - the gene drive cassette - to be copied onto the previously unaltered chromosome. With two versions of the drive cassette, the organism is certain to pass it on to all of its offspring.  See the gene drives page for pictures and additional details.

Cas9 can also be used to regulate biological systems, which by and large operate through selective stickiness. Molecules that stick together are more likely to continue to interact and influence one another's activity. Since Cas9 is a protein that can sequence-specifically bind RNA and DNA molecules, it can selectively interact with all three major types of biopolymers in the cell. This means it can be used to recruit regulatory proteins or RNAs to any DNA sequence in order to control gene expression, among many other uses. Using orthogonal Cas9 proteins that don't recognize the same guide RNAs allows different types of molecules to be localized to different sequences within the same cell, enabling some genes to be activated while others are repressed and still more targeted for degradation.

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