Introduction Heterologous protein expression underpins many advancements in modern molecular biology, including protein engineering, production of bioindustrial enzymes and therapeutics, and structure-function discovery. Heterologous proteins are commonly expressed from plasmids to facilitate cloning and subsequent genetic manipulation. However, plasmid-based expression has many drawbacks, including reduced stability, population drift, and limited control over copy number variations. Combining CRISPR editing tools (1, 2) and advancements in gene sequence engineering (3, 4), heterologous gene integration into chromosomal DNA has become more accessible, offering genomic stability with consistent, high expression (5). These advances facilitate in-depth examination of sequence-function relationships and systematic control of protein activity from the genome. Thorough interrogation of gene sequence variants remains difficult, even across a single locus. To do this efficiently and cost-effectively, libraries of genetic edits must be designed and generated in multiplex, which necessitates trackability of designs to connect phenotype to genotype. An innovative genome engineering technology - the Onyx™ Digital Genome Engineering Platform - performs genome-wide and trackable CRISPR editing at scale in an automated benchtop device. This approach significantly reduces the time and resources spent on constructing variant libraries, as demonstrated in this app note. Here we used Inscripta’s open-sourced MAD7TM CRISPR nuclease to integrate a green fluorescent protein (GFP) gene into the E. coli MG1655 chromosome. We then used the Onyx platform to design and generate GFP site-saturation libraries, with 723 designs targeting functional residues intended to modify fluorescence intensity and spectral characteristics (Figure 1A). Additionally, we generated a library of 8,284 distinct promoter and ribosome binding site (RBS) sequences extracted from the E. coli genome, inserted it 5’ of the GFP gene and screened this library to quantify the strength of native E. coli promoters and RBS sites in a massively parallel experiment.
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