Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

Abstract

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

gscreend: modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection

“…We hypothesize that the broadening of library distribution is due to sequence-specific differences in synthesis or amplification efficiency. A recently published approach to synthesize covalently-closed-circular-synthesized (3Cs) gRNA libraries may thus be a promising technology for substantial reduction of library width and experiment size [52].…”

Integrative analysis of epigenetics data identifies gene-specific regulatory elements

“…We use a recently published genome-wide doxorubicin CRISPR-Cas9 resistance screen [56] to show another possible application of StitchIt, which is the association of experimentally highlighted regions to their most likely target genes. As stated above, the CRISPR-Cas9 screen led to 332 non-coding target sites of 226 validated gRNA sequences.…”

“…Here, we use a genome-wide CRISPR perturbation library consisting of partially randomized degenerated oligonucleotides (5'-NNDNNNNNHNNNNHDHNVVR-3') with flanking 3Cs homology regions which was created using ssDNA of template-plasmids and site-specific mutagenesis targeting coding and non-coding regions of the human genome in hTERT-RPE1 cells from ATCC (CRL-4000) [56]. In total, 226 unique gRNAs could be mapped to the coding and non-coding part of the genome, resulting in 332 unique genomic target sites.…”

multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets

“…A further example for a recently emerged Crispr/Cas9 application is parallel targeting of many loci with gRNA libraries, required for instance when targeting transcription factor binding sites (TFBS) or their neighborhoods (Shariati et al 2019). Other screening oriented Crispr/Cas9-based applications include genome wide visualization , or complex gRNA libraries to investigate cell fitness (Wegner et al 2019). For such applications, the total number of gRNAs, or library complexity, directly correlates with effort and costs.…”