CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries

Abstract

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 wet-lab 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 distinct 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 resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.

A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation

“…Additionally, we know from our examination of ARRB2/Arrestin3 that our selected FDR<0.05 excluded true positive hits, and thus it is probable additional candidate genes exist in our data beyond to the 492 highlighted here. One approach that could address this challenge is to utilize small, custom libraries consisting of hundreds of sgRNAs to be analyzed in a pooled format (Nagy and Kampmann 2017; Becker et al 2020). There are several advantages to such an approach: 1) a reasonably compact micro-library of hits could be analyzed with high coverage (>1000x), allowing greater sensitivity to small effects; 2) the reduced number of sgRNAs compared to the genome-level would allow multiple conditions to be tested in parallel in a reasonable time frame.…”

CRISPR/Cas9 screens implicate RARA and SPNS1 in doxorubicin cardiotoxicity

“…The targeted library comprised 7820 sgRNAs targeting 952 genes (8 sgRNAs per gene) and 200 non-targeting sgRNAs. Oligonucleotides were synthesized (Agilent) and Gibson-cloned into lentiCRISPR v2 and analyzed as described previously (62,63). Briefly, library coverage (99%) and sgRNA skew factor (4.3) were well within recommended limits.…”