Using a combination of biochemical, structural, and genetic approaches, we found that Cas1 and Cas2 functions as a protein complex.
The Cas1–Cas2 complex captures ~30 bp of foreign DNA and integrates them into the CRISPR locus via a direct nucleophilic reaction similar to many retroviral integrases and transposases.
In this review, we attempt to give a concise overview of recent progress made in mechanistic understanding of protein aggregation, particulate formation and protein solution rheology.
CRISPR-harboring organisms generate immunological memory of previous infections by capturing short segments of foreign DNA for integration into CRISPR loci as spacer sequences.
Central to this process are Cas1 and Cas2 – the only conserved proteins in all CRISPR systems.
In collaboration with Eric Greene’s laboratory at Columbia University, we have applied a combination of single-molecule and bulk biochemical experiments to resolve the mechanism of DNA interrogation for two phylogenetically unrelated complexes: Cas9, the DNA-targeting protein found in Type II CRISPR–Cas systems (S.
pyogenes), and Cascade, the DNA-targeting complex found in Type I-E CRISPR–Cas systems (E. Our results have revealed that the target search is PAM-guided, and that these distinct RNA-guided complexes have converged on a common mechanism for target DNA recognition.
The focus of the review is primarily on biotherapeutics and proteins/antibodies that are relevant to that area.
As per the remit of , here we attempt to stimulate interest in areas of debate.
While the field is certainly not mature enough that all problems may be considered resolved and accepted by consensus, we wish to highlight some areas of controversy and debate that need further attention from the scientific community.
The development of stable protein-based formulations with controlled rheological response is an area of high interest for the high-growth biotherapeutic industry, as well as for more traditional industrial sectors such as foods.
Although the final applications in these two industrial sectors are very different, the complex self-assembly and particle formation processes under various formulation conditions (p H, ionic strength, buffer salts, temperature) must be well-understood, characterized, and controlled.
This then allows the development of formulations which remains stable with long shelf life and that exhibits rheological properties that enhance/optimize the application performance — e.g.
Cascade is composed of Cse1, Cse2, Cas7, Cas5e, and Cas6e subunits and one cr RNA, forming a structure that binds and unwinds ds DNA to form an R-loop.