This invention enables target specific DNA destruction in a highly controlled and regulated manner.
Current methods for removing genetic material from a living prokaryotic host incompletely removes the genetic element from the population at large. Furthermore, targeting specificities of all other known prokaryotic nucleases capable of degrading DNA in vivo are encoded in their protein sequences, making it difficult if not impossible to readily alter the intended target sequence specificity without extensive mutagenesis and screening. This invention is a DNA destruction device (DDD) that uses CRISPR biology to address both of these issues; it prevents propagation of a DNA sequence to progeny while actively degrading these elements, lowering the total copy number of existing elements in the population gene pool and can be simply and quickly re-targeted to destroy specific DNA.
The device is composed of two essential components engineered from E. coli genes. The first is the CRISPR array, a synthetic DNA sequence containing series of unique 32-bp fragments interspersed between identical 29-bp palindromic repeats of specific sequence. The array's spacers encode the device's targeting information. The second component is a cas gene (cas3 or casABCDE), which encodes the device's actuator functions, specifically the enzymatic machinery sufficient for catalyzing DNA degradation. Both of these essential components are put under the artificial control of independent inducible promoters, which allow for user controlled activation of the device in response to specific combination of environmental or chemical signals. The device is highly effective at removing specific genetic elements from cells when and where desired. Some examples of targeted genetic elements could be exogenous dangerous or confidential DNA sequences or DNA being used by the host cell for protein or other production.
Allows for the user-controlled, irreversible removal
of virtually any genetic element