The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wetlab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www. crispr-clue.de. All in all, CLUE represents a resourcesaving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.
GrantsDr Helmut Legerlotz Stiftung Care for Rare Foundation Beug Foundation for Metastasis Research Society for the Advancement of Science and Research of the LMU Medical Faculty (WiFoMed) European Research Council Mildred Scheel Professorship by German Cancer Aid German Research Foundation (DFG) Collaborative Research Center 1243 `Genetic and Epigenetic Evolution of Hematopoietic Neoplasms' DFG Bettina Brau Stiftung Max-Eder Program of Deutsche Krebshilfe