SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries

Abstract

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled innovative CRISPR-based methods, such as the visualization of chromatin dynamics in living cells. These libraries have the potential to be applied to a vast number of biological systems and aid in the development of new technologies, but their synthesis is hindered by the cost, time requirements, and technical difficulty of current sgRNA library generation methods. Here, we describe SLALOM—a rapid enzymatic method for generating robust, variant-matched sgRNA libraries from any source of DNA in under 3 hours. This method utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports using a strand displacing polymerase. Using this method, we have constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its potential to expand the reach of CRISPR technology and facilitate methods requiring custom sgRNA libraries.