Related Experiment Video
Updated: May 15, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
A CRISPR-Cas9 System for Knock-out and Knock-in of High Molecular Weight DNA Enables Module-Swapping of the
Zhe-Chong Wang1, Hayden Stegall1, Takeshi Miyazawa1
1The University of Texas at Austin.
Background:
Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.
Results:
We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.
Conclusion:
Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.
Related Concept Videos
CRISPR
Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
In-vitro Mutagenesis
CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

