CRISPR-Cas9 Part II

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(New page: <StructureSection load='' size='350' side='right' scene='74/742625/Cv3/12' caption=''> ====Crystal Structure of Staphylococcus aureus Cas9<ref name="P5CZZ">PMID:26317473</ref>==== The RN...)
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toolbox, as we have done previously using the SpCas9 crystal structure. Given the similarities in the overall domain organizations of SaCas9 and SpCas9, we initially explored the feasibility of engineering the SaCas9 sgRNA, to develop robust transcription activators. In the SpCas9 structure, the tetraloop and stem loop 2 of the sgRNA are exposed to the solvent, and permitted the insertion of RNA aptamers into the sgRNA to create robust RNA-guided transcription activators. To generate the SaCas9-based activator system, a catalytically inactive version of SaCas9 (dSaCas9) was created by introducing the D10A and N580A mutations to inactivate the RuvC and HNH domains, respectively, and attached VP64 to the C terminus of dSaCas9. The sgRNA scaffold was modified by the insertion of the MS2 aptamer stem loop (MS2-SL), to allow the recruitment of MS2-p65-HSF1 transcriptional activation modules. To evaluate the dSaCas9-based activator design, a transcriptional activation reporter system was constructed, consisting of tandem sgRNA target sites upstream of a minimal CMV promoter driving the expression of the fluorescent reporter gene mKate2. An additional transcriptional termination signal upstream of the reporter cassette was included, to reduce the background previously observed in a similar reporter. Robust activation of mKate2 transcription was observed when the engineered sgRNA complementary to the target sites was expressed, whereas the non-binding sgRNA had no detectable effect. Based on a screening of different sgRNA designs with this reporter assay, was found that the insertions of MS2-SL into the tetraloop and putative stem loop 2 induced strong activation in our reporter system, whereas the insertion of MS2-SL into stem loop 1 yielded modest activation, consistent with the structural data. The single insertion of MS2-SL into the tetraloop was the simplest design that yielded strong transcriptional activation. Using this optimal sgRNA design, we further tested the activation of endogenous targets in the human genome. Two guides were selected each for the human ASCL1 and MYOD1 genomic loci, and demonstrated that the dSaCas9-based activator system activated both genes to levels comparable to those of the dSpCas9-based activator. Given that the sgRNAs for SaCas9 and SpCas9 are not interchangeable, the SaCas9-based transcription activator platform complements the SpCas9-based activator systems, by allowing the independent activation of different sets of genes. The SpCas9 structure also facilitated the rational design of split-Cas9s, which can be further engineered into an inducible system. This SaCas9 structure revealed several flexible regions in SaCas9 that could likewise serve as potential split sites. Three versions of a split-SaCas9 were created, and two of them showed robust cleavage activity at the endogenous EMX1 target locus. Using the best split design, inducible schemes were then tested based on the abscisic acid (ABA) sensing system, as well as two versions of the rapamycin-inducible FKBP/FRB system. All three systems were able to support inducible SaCas9 cleavage activity, demonstrating the possibility of an inducible, split-SaCas9 design; however, further optimization is required to increase its efficiency and reduce its background activity.
toolbox, as we have done previously using the SpCas9 crystal structure. Given the similarities in the overall domain organizations of SaCas9 and SpCas9, we initially explored the feasibility of engineering the SaCas9 sgRNA, to develop robust transcription activators. In the SpCas9 structure, the tetraloop and stem loop 2 of the sgRNA are exposed to the solvent, and permitted the insertion of RNA aptamers into the sgRNA to create robust RNA-guided transcription activators. To generate the SaCas9-based activator system, a catalytically inactive version of SaCas9 (dSaCas9) was created by introducing the D10A and N580A mutations to inactivate the RuvC and HNH domains, respectively, and attached VP64 to the C terminus of dSaCas9. The sgRNA scaffold was modified by the insertion of the MS2 aptamer stem loop (MS2-SL), to allow the recruitment of MS2-p65-HSF1 transcriptional activation modules. To evaluate the dSaCas9-based activator design, a transcriptional activation reporter system was constructed, consisting of tandem sgRNA target sites upstream of a minimal CMV promoter driving the expression of the fluorescent reporter gene mKate2. An additional transcriptional termination signal upstream of the reporter cassette was included, to reduce the background previously observed in a similar reporter. Robust activation of mKate2 transcription was observed when the engineered sgRNA complementary to the target sites was expressed, whereas the non-binding sgRNA had no detectable effect. Based on a screening of different sgRNA designs with this reporter assay, was found that the insertions of MS2-SL into the tetraloop and putative stem loop 2 induced strong activation in our reporter system, whereas the insertion of MS2-SL into stem loop 1 yielded modest activation, consistent with the structural data. The single insertion of MS2-SL into the tetraloop was the simplest design that yielded strong transcriptional activation. Using this optimal sgRNA design, we further tested the activation of endogenous targets in the human genome. Two guides were selected each for the human ASCL1 and MYOD1 genomic loci, and demonstrated that the dSaCas9-based activator system activated both genes to levels comparable to those of the dSpCas9-based activator. Given that the sgRNAs for SaCas9 and SpCas9 are not interchangeable, the SaCas9-based transcription activator platform complements the SpCas9-based activator systems, by allowing the independent activation of different sets of genes. The SpCas9 structure also facilitated the rational design of split-Cas9s, which can be further engineered into an inducible system. This SaCas9 structure revealed several flexible regions in SaCas9 that could likewise serve as potential split sites. Three versions of a split-SaCas9 were created, and two of them showed robust cleavage activity at the endogenous EMX1 target locus. Using the best split design, inducible schemes were then tested based on the abscisic acid (ABA) sensing system, as well as two versions of the rapamycin-inducible FKBP/FRB system. All three systems were able to support inducible SaCas9 cleavage activity, demonstrating the possibility of an inducible, split-SaCas9 design; however, further optimization is required to increase its efficiency and reduce its background activity.
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=See aslo=
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*[[Cas9]]
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*[[Endonuclease]]
</StructureSection>
</StructureSection>
== References ==
== References ==
<references/>
<references/>
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[[Category:Topic Page]]
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[[Category:Crispr]]
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[[Category: Crispr-associated]]
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[[Category: endonuclease]]
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[[Category: Cas9]]

Revision as of 13:36, 2 September 2018

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References

  1. Nishimasu H, Cong L, Yan WX, Ran FA, Zetsche B, Li Y, Kurabayashi A, Ishitani R, Zhang F, Nureki O. Crystal Structure of Staphylococcus aureus Cas9. Cell. 2015 Aug 27;162(5):1113-26. doi: 10.1016/j.cell.2015.08.007. PMID:26317473 doi:http://dx.doi.org/10.1016/j.cell.2015.08.007

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Alexander Berchansky

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