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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.
Mechanisms by which specific histone modifications regulate distinct gene regulatory networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression in mammalian cardiogenesis. Early embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific gene regulatory networks at two critical cardiogenic junctures: left ventricle patterning and postnatal cardiomyocyte cell cycle withdrawal. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, these analyses also revealed that H3K79me2 in specific regulatory elements contributed to silencing genes usually not expressed in cardiomyocytes. As DOT1L mutants had increased numbers of postnatal mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity, controlled inhibition of DOT1L might be a strategy to promote cardiac regeneration post-injury.