Our Design Process
In selecting a gene elimination mechanism, we identified the most important aspects: minimal burden on cell growth and the ability to be delivered and propagated. We strayed away from some systems, such as Cas9, due to the toxicity in cells that lack efficient non-homologous end joining repair mechanisms, and had difficulty in propagating throughout a bacterial population . Instead, we pursued the use of the INTEGRATE system, a CRISPR-guided transposase capable of highly specific, autonomous gene elimination .
What is INTEGRATE?
INTEGRATE is a one-plasmid system that contains all the necessary molecular machinery for targeted DNA insertions into sequence specific loci . INTEGRATE is a CRISPR-guided DNA integration system composed of the CRISPR-associated proteins Cas6, Cas7, and Cas8(also referred to here as Cas5), which come together to form the