SLALOM: A Simple and Rapid Method for Enzymatically Generating CRISPR-Cas9 sgRNA Libraries
CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled the proliferation of several innovative CRISPR-based methods. As these libraries find their way into experiments involving non-model organisms or new molecular techniques, the need to create libraries targeting custom sets of genes is increasing. Here, we describe SLALOM--a rapid enzymatic method for generating robust, variant-matched sgRNA libraries from any DNA source. This method utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports using a strand displacing polymerase to create a workflow consisting of five steps conducted in a single reaction vessel in under 3 hours. Using this method, we have constructed libraries from the E. coli genome and the transcriptome of developing zebrafish hearts. This method will expand the reach of CRISPR technology to non-standard organisms and facilitate methods requiring highly customizable sgRNA libraries.