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The rod-shaped filamentous phage M13 differs from other bacteriophages in that its genome-packaging capacity is variable and in that it is present as single-stranded (ss) DNA.
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CRISPR libraries have become ubiquitously used in functional genomics efforts, underscoring relevance and utility of new PCR- and cloning-free technologies. Owing to these technical constraints, conventional libraries contain an unwanted PCR and cloning-dependent bias in their gRNA distribution that influences the experimental scale required for statistically significant hit calling ( Shalem et al., 2014 Wang et al., 2014). Conventionally used methods to generate gRNA libraries in pooled or arrayed formats include T4 ligase or homology-based cloning techniques, which require the PCR-based amplification of gRNA-encoding oligonucleotides as well as the presence of open plasmid DNA for successful gRNA sequence cloning ( Arakawa, 2016 Koike-Yusa et al., 2014 Ong et al., 2017 Schmidt et al., 2015 Shalem et al., 2014 Vidigal and Ventura, 2015 Wang et al., 2014). A fundamentally important aspect of high-fidelity CRISPR/Cas screening is the quality of the gRNA library that is interrogated, with its diversity and distribution primarily influencing downstream experimental scales ( Sanson et al., 2018). 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.ĬRISPR/Cas has rapidly become the gold standard for unbiased high-throughput experiments, outperforming preexisting technologies such as RNAi ( Evers et al., 2016 Morgens et al., 2016). 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. 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. 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. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps.