MICROBIAL CELL ENGINEERING

 

Engineering microbial cells that produce high amounts of heterologous protein can be difficult.

We are currently developing a genomic screening platform that can be used to test thousands of genomic modifications simultaneously to quickly generate data that can be used as a roadmap for increasing expression of a target protein in a specific cell type.

With this new platfrom companies can achieve faster development times, better bioprocesses, and strains that outcompete other players in the industry.

 

CRISPR LIBRARY GENERATION

 

SLALOM 1.0™ sgRNA Library Synthesis Kits, S. pyogenes provide a simple and quick method for enzymatically synthesizing high quality sgRNA libraries through a sequence of fast reactions, using the supplied reagents and source of DNA supplied by the user.

The CRISPR Cas9 complex from S. pyogenes can be programmed to bind to a specific DNA sequence by simply altering the first 20 nucleotides of a single guide RNA (sgRNA)¹. Libraries of sgRNA molecules have enabled new techniques including live cell chromatin imaging and functional genomic screening, but the synthesis of custom libraries can often be a bottleneck in these experiments. SLALOM 1.0 ™ sgRNA Library Synthesis Kits permit the synthesis of a custom library in a few hours and without specialized bioinformatic techniques.

SLALOM 1.0™ sgRNA Library Synthesis Kits, S. pyogenes are based on SLALOM (sgRNA Library Assembly by Ligation Onto Magnetic beads)², which works on the principle of eznymatically extracting valid spacer sequences from a source of DNA. These kits were designed to be quick and highly efficent and include optional control DNA and a troubleshooting guide to ensure fast synthesis of custom libraries.

 

 

Figure 1: Workflow of SLALOM 1.0 ™ sgRNA Library Synthesis Kits, S. pyogenes.

(A). A source of DNA such as Genomic DNA or cDNA is isolated. (B). The DNA source is enzymatically digested at protospacer adjacent motifs (PAMs) to create DNA fragments with valid spacer sequences on each end of the fragments. (C). The SLALOM 1.0™ Capture Adapter is ligated to the ends of the fragments. (D). Using a restriction enzyme that cleaves approximately 20 bp away from its reqconition sequence, the spacer sequences are captured and the remaining fragments are washed away using bead purification. (E). A promoter or cloning adapter is ligated to the spacer sequence. (F). The final library is detached from the beads and nicks in the library are repaired. The library can be used as a template for in vitro transcription or cloned into plasmids depending on the downstream application.

 

References

1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.

2. Yates JD, Russell RC, Barton NJ, Yost HJ, Hill JT. A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries. Nucleic Acids Res. 2021;49(22):e131.