Difference between revisions of "Part:BBa K4630201"

Latest revision as of 17:09, 11 October 2023

The optimized pCas

CRISPR technique is a powerful tool for genome engineering, but its efficiency and precision are sometimes suboptimal. Our design, CRISPReporter, requires a highly efficient editing method to ensure the sensitivity and accuracy of the cascade recording system. Based on a previous study, we adopted a one-plasmid system which we called pCas (pRed-Cas9-poxb300) that combines Cas9 and Lambda-Red recombinases in E. coli, which reported to achieve 100% genome editing efficiency in 6 hours of induction.1 We constructed this new composite part from the plasmid pCas, which we obtained from Genescript.2 In this case, the pCasop (short for pCas_optimized) plasmid suits our project better.

This new composite part consists of a Cas9 coding sequence (BBa_K1218011), and a Lambda-Red recombinases coding sequence (BBa_K1433005). The expression of Cas9 and Lambda-Red is under the control of araBAD promoter. We demonstrated that this one plasmid editing system can perform both genome and plasmid editing, with high editing efficiency and no mutations.

Sequence and Features