Since its inception, CRISPR-Cas technology has evolved from a revolutionary breakthrough to a versatile tool in genetic engineering. Researchers have continued to finetune this technology, improving all aspects of its application, from ease of application and expanded species compatibility to enhanced accuracy and coverage. The latest of these improvements, published in Nucleic Acids Research, assures that this upward trajectory will not be slowing down any time soon. Aptly named "pAblo·pCasso," this novel CRISPR-Cas toolkit, developed by researchers at The Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain), expands the spectrum of genome sites available for base-editing, propelling the rapid evolution of bacteria for a myriad of bioproduction applications. 

The emergence of 'pAblo·pCasso' sets a new benchmark in the realm of CRISPR-Cas technologies. It is able to execute precise and reversible DNA edits within Gram-negative bacteria—a feat that remained elusive with earlier CRISPR systems. Postdocs Dr. Ekaterina Kozaeva and Manuel Nieto-Domínguez emphasized, “Tools such as the pAblo·pCasso plasmids redefine our approach to genetic manipulation of bacteria, particularly those atypical ones that pose greater engineering challenges." This toolkit employs specialized fusion enzymes, modified Cas9 in tandem with editor modules CBE or ABE. These modules act as ‘molecular pencils,’ meticulously altering specific DNA nucleotides to ensure exceptional accuracy in controlling gene function.

"A major innovation of this approach lies in its capacity to access and engineer previously untapped sites for genome editing, leaving no traces behind,” explained Kozaeva and Nieto-Domínguez. Traditional CRISPR-Cas systems depend on specific DNA sequences (PAM sequences) near the target site for recognition and are less effective at making precise single-nucleotide changes. 'pAblo·pCasso,' however, eliminates the need for specific PAM sequences by integrating advanced Cas-fusion variants that do not require these sites, greatly expanding the range of possible targets. Furthermore, its reversible editing capability, facilitated by specialized enzymes, permits temporary modifications—an imperative feature for dynamic and controlled gene studies.

This technology is set to significantly amplify the capabilities of researchers and industries engaged in engineering bacterial cell factories. Kozaeva and Nieto-Domínguez emphasized, “We can now fabricate bacterial cell factories within days rather than months!”.  By facilitating swift and precise genetic modifications, 'pAblo·pCasso' propels the development of bacteria across a diverse array of bioproduction applications, spanning from pharmaceuticals to biofuels. This alignment with sustainable production goals positions 'pAblo·pCasso' as a promising new player in reshaping the landscape of genetic engineering. 

"With pAblo·pCasso, we've shattered the barriers of traditional CRISPR technology. This toolkit unfurls new possibilities for bacterial engineering, steering us towards efficient and sustainable bioproduction with engineered bacteria," stated DTU Biosustain's Professor Pablo I. Nikel.