Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • In this study we describe an efficient dCas activator

    2018-10-20

    In this study, we describe an efficient dCas9 activator with multimeric VP16 activation domain and a simplified method for guide RNA assembly and cloning. We demonstrate that the dCas9 activator can be fused to a dihydrofolate reductase (DHFR)-derived destabilization domain (DD) (Iwamoto et al., 2010) and used to control gene poly ic Supplier with addition of Trimethoprim (TMP) in multiple human cell types. Furthermore, we demonstrate that the dCas9 activator can be used to replace transgenic OCT4 in human cell reprogramming and that human pluripotent cell differentiation can be induced by the activator in a TMP-dependent manner.
    Results
    Discussion Our results show that increasing the number of VP16 transactivator repeats fused to dCas9 leads to improved activation of the endogenous OCT4. Using dCas9VP192 and five gRNAs, we obtained up to 70-fold OCT4 upregulation in HEK293 cells, in similar range of efficiency with previous studies (Cheng et al., 2013; Hu et al., 2014; Mali et al., 2013b). The fact that dCas9 activators can replace transgenic OCT4 as a reprogramming factor in human iPSC derivation speaks for the functional relevance of the gene activation and the potential for utilizing this approach for human cell type conversions. A similar approach has been previously demonstrated to work for mouse cell reprogramming (Chakraborty et al., 2014; Gao et al., 2014). The potency of dCas9 activators varies between genes. We were not able to efficiently replace additional reprogramming factors, although their activation could be detected in fibroblasts. As NANOG and LIN28A activation was detected only in a minor population of cells, it is likely that the activation of these genes is stochastic by nature and limited by epigenetic barriers. Therefore, alternative approaches, for example, improved transactivation domains or catalytically active domains affecting the epigenetic state of the gene regions may be needed (Chavez et al., 2015; Hilton et al., 2015; Konermann et al., 2015; Maeder et al., 2013b). Additionally, the size of the construct could be reduced by incorporating multimerization sites for the activator domains, such as the recently reported SunTag peptide tail, which would implement an amplification step in the recruitment of transactivators to the proximal promoter that results in greatly enhanced gene activation (Tanenbaum et al., 2014). Targeting noncoding RNAs as well as genomic enhancer elements in addition to promoter proximal areas might also help in replacing transgenic factors with only dCas9 activators and gRNAs (Gao et al., 2014; Hilton et al., 2015). The power of the dCas9 system for affecting gene expression lies in the multiplexing potential of the short gRNA sequences. Concatenation of gRNA expression cassettes using Golden Gate cloning has recently been described for multiplex genome editing (Sakuma et al., 2014) and gene activation (Kabadi et al., 2014). In contrast, our method avoids time-consuming traditional cloning for the initial gRNA-assembly step, being substituted by a simple PCR reaction. After gRNA efficiency validation, the gRNA-PCR fragments are concatenated with a single Golden Gate reaction, streamlining the generation of gRNA-encoding plasmids for different target genes. The small size of the gRNA construct compared with transgenic expression methods allows for efficient simultaneous targeting of multiple genomic loci and potentially full gene regulatory networks (GRNs). As dCas9 activators are targeting the endogenous genes for activation, activation of the proximal regulatory regions might benefit the establishment of proper transcriptional programs by affecting epigenetic barriers to the reprogramming process. N-terminal fusion of the dCas9 activator with the DHFR DD leads to a functional TMP-dependent dCas9 activation. In terms of inducible gene transcription, DDdCas9 activator performs as efficiently as the TetON dCas9, with the advantage of not requiring an additional rtTA transactivator element. The DDdCas9 activator did, however, show a certain level of leakiness possibly due to incomplete protein degradation. This nonspecific activation can be controlled by combining the DDdCas9 activator with additional inducible systems like TetON, as shown in Figure 3B, or by controlling the expression level of the activator. Moreover, combining the S. pyogenes DDdCas9 activator with other orthogonal Cas9-derived conditional transcriptional modulators should allow for simultaneous temporal control over multiple gene expression programs (Davis et al., 2015; Esvelt et al., 2013; Ran et al., 2015; Zetsche et al., 2015).