Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression.
The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.
We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of changes to the Cas9 protein in addition to the fusion of a transcriptional activation domain (AD) [1][2][3][4][5] . However, the modest levels of gene activation achieved by current Cas9 activators have limited their potential applications. Here we describe the development of an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of induced pluripotent stem cells (iPSCs).
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins offer a breakthrough platform for cheap, programmable, and effective sequence-specific DNA targeting. The CRISPR-Cas system is naturally equipped for targeted DNA cutting through its native nuclease activity. As such, groups researching a broad spectrum of biological organisms have quickly adopted the technology with groundbreaking applications to genomic sequence editing in over 20 different species. However, the biological code of life is not only encoded in genetics but also in epigenetics as well. While genetic sequence editing is a powerful ability, we must also be able to edit and regulate transcriptional and epigenetic code. Taking inspiration from work on earlier sequencespecific targeting technologies such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs), researchers quickly expanded the CRISPR-Cas toolbox to include transcriptional activation, repression, and epigenetic modification. In this review, we highlight advances that extend the CRISPR-Cas toolkit for transcriptional and epigenetic regulation, as well as best practice guidelines for these tools, and a perspective on future applications.
Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.
Here we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single nucleotide polymorphism. We employ our methodology to generate a novel in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.Precise Cas9 targeting enables genomic mutation prevention | bioRχiv |
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