Recombineering machinery to increase homology directed genome editing in thermophilic microbes

Patent · Tue Jan 23 04:00:00 EST 2024

OSTI ID:2541706

Eckert, Carrie; Walker, Julie E.

A native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat/cas systems were developed and characterize to improve the ability to engineer C. thermocellum and other thermophilic microbes. The native Type I-B system was engineered for genome editing. For the Type I-B system, an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus. When recombineering machinery was expressed the efficiency was increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed. When recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, a new tool was developed that allows for efficient CRISPR editing. The tools provided herein enable CRISPR technologies to better engineer C. thermocellum, including engineering C. thermocellum for both increased lignocellulose degradation and biofuel production.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); University of Colorado, Denver, CO (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-00OR22725
• Assignee: The Regents of the University of Colorado, a body corporate (Denver, CO)
• Patent Number(s): 11,879,134
• Application Number: 17/012,698
• OSTI ID: 2541706
• Country of Publication: United States
• Language: English