CRISPR Technologies 2.0: Harnessing the Next Generation of Cas Nucleases for Genome Editing and Beyond Conference: Synthetic Biology Engineering Evolution Design SEEDYear: 2017Proceeding: 2017 Synthetic Biology: Engineering, Evolution & Design (SEED)Group: General SubmissionsSession: Session 9: Genome Editing Time: Thursday, June 22, 2017 - 2:00pm-2:30pm Authors: Beisel, C. L., North Carolina State University CRISPR technologies represent revolutionary tools for gene editing in basic research, biotechnology, medicine, and agriculture and have driven ethical debates around GMOâs and editing human germline cells. Despite the fanfare surrounding these technologies, they have a common, natural source: adaptive immune systems in bacteria and archaea called CRISPR-Cas systems. CRISPR-Cas systems use guide RNAs to direct Cas nucleases to bind and cleave complementary genetic material. Guide RNAs can be readily designed de novo to direct the binding and cleavage of specific DNA sequences, lending to gene editing and many other applications. While existing CRISPR technologies have overwhelmingly relied on the Cas9 nuclease, nature offers a myriad of other Cas proteins with unique mechanisms and functions. Harnessing these more recently discovered nucleases holds the potential to improve and expand existing CRISPR technologies and bring about a further biotechnological revolution. Here, I will discuss my groupâs work on the recently discovered Type V-A Cas nuclease Cpf1. Like the standard S. pyogenes Cas9, Cpf1 is a single effector protein that is directed by guide RNAs to cleave target DNA. However, unlike Cas9, Cpf1 is smaller, generates an overhang cleavage product, and can process a native CRISPR array into individual guide RNAs without any accessory factors. These features could allow Cpf1 to eventually displace Cas9 across its many uses. We have been rapidly prototyping Cpf1 and other novel Cas nucleases using cell-free TXTL systems, generating design rules for genome editing and programmable gene regulation. We have also developed an efficient, one-pot cloning scheme for large CRISPR arrays for multiplexing applications. These advances are transitioning Cpf1 and other recently discovered Cas nucleases into a new generation of CRISPR technologies for emerging applications in genome editing, scalable gene circuit design, and high-throughput phenotypic screens.