A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing

Liu, Kaiwen Ivy; Ramli, Muhammad Nadzim Bin; Woo, Ariel; Wang, Yuanming; Zhao, Tianyun; Zhang, Xiujun; Yim, Daniel; Chong, Bao Yi; Ali, Gowher; Chua, Mervyn Zi Hao; Jung, Jonathan; Lee, Jia Hui Jane; Tan, Meng How

doi:10.1038/nchembio.2179

Cited by 186 publications

(178 citation statements)

References 35 publications

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“…On the other hand, lessening the promiscuity of SpCas9 can be achieved by lowering its activity on off-target sequences while maintaining reasonably effective on-target activities. This is attempted by minimizing the exposure of the DNA to SpCas9 activity by limiting the time of its expression [24,25,[54][55][56][57] or decreasing the level of protein [58,59], employing modified sgRNAs (truncated [23] or 5′ extended with a GG dinucleotide [21]) or weakening the protein-DNA interactions [32,33]. Although a systematic comparison of these approaches is still missing, they have led to varying success and it has been observed that different targets are cleaved with different off-target propensity by SpCas9.…”

Section: Discussionmentioning

confidence: 99%

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

et al. 2017

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Background: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. Results: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5′ G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.

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Section: Discussionmentioning

confidence: 99%

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

et al. 2017

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“…Manipulation at the transcriptional level, however, offers poor temporal control compared with post-transcriptional and post-translational control, and is difficult to achieve in many cell types due to a lack of tissue-specific promoters. Post-translationally regulated Cas9 variants have been developed in which conditionally inactive Cas9 proteins are expressed and genome editing activity is restored only in the presence of specific stimuli12131415. Controlling the activity of Cas9 to limit the temporal exposure of cells to genome editing activity has resulted in important benefits such as reduced modification of off-target loci13.…”

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confidence: 99%

Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

2017

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Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

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“…To limit Lama1 expression only to skeletal muscles, the promoter of deSaCas9-VP160 should be muscle specific. Moreover, to prevent overexpression of Lama1, the Cas9-VP64 gene or the sequences coding for the sgRNAs can be placed under a promoter inducible by doxycycline and 4-hydroxytamoxifen 41, 42, 43, 44…”

Section: Discussionmentioning

confidence: 99%

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160

Perrin

Rousseau

Tremblay

2017

Molecular Therapy - Nucleic Acids

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Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies.

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A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing

Cited by 186 publications

References 35 publications

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160

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