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cleaves the non-target DNA strand through a two-metal ion mechanism, as in other RNase H superfamily endonucleases. The HNH domain of SaCas9 has a ββα-metal fold, and shares structural similarity with those of SpCas9 (27% identity, rmsd of 1.8 A˚ for 93 equivalent Ca atoms) and AnCas9 (18% identity, rmsd of 2.6 A˚ for 98 equivalent Ca atoms). Asp556, His557, and Asn580 of SaCas9 are located at positions similar to those of the catalytic residues of SpCas9 (Asp839, His840, and Asn863) and AnCas9 (Asp581, His582, and Asn606). Indeed, the H557A and N580A mutants of SaCas9 almost completely lacked DNA cleavage activity, suggesting that the SaCas9 HNH domain cleaves the target DNA strand through a one-metal ion mechanism, as in other ββα-metal endonucleases. A structural comparison of SaCas9 with SpCas9 and AnCas9 revealed that the RuvC and HNH domains are connected by α-helical linkers, L1 and L2, and that notable differences exist in the relative arrangements between the two nuclease domains. A biochemical study suggested that PAM duplex binding to SpCas9 facilitates the cleavage of the target DNA strand by the HNH domain. However, in the PAM-containing quaternary complex structures of SaCas9 and SpCas9, the HNH domains are distant from the cleavage site of the target DNA strand. A structural comparison of SaCas9 with Thermus thermophilus RuvC in complex with a Holliday junction substrate indicated steric clashes between the L1 linker and the modeled non-target DNA strand, bound to the active site of the SaCas9 RuvC domain. These observations suggested that the binding of the non-target DNA strand to the RuvC domain may facilitate a conformational change of L1, thereby bringing the HNH domain to the scissile phosphate group in the target DNA strand.
cleaves the non-target DNA strand through a two-metal ion mechanism, as in other RNase H superfamily endonucleases. The HNH domain of SaCas9 has a ββα-metal fold, and shares structural similarity with those of SpCas9 (27% identity, rmsd of 1.8 A˚ for 93 equivalent Ca atoms) and AnCas9 (18% identity, rmsd of 2.6 A˚ for 98 equivalent Ca atoms). Asp556, His557, and Asn580 of SaCas9 are located at positions similar to those of the catalytic residues of SpCas9 (Asp839, His840, and Asn863) and AnCas9 (Asp581, His582, and Asn606). Indeed, the H557A and N580A mutants of SaCas9 almost completely lacked DNA cleavage activity, suggesting that the SaCas9 HNH domain cleaves the target DNA strand through a one-metal ion mechanism, as in other ββα-metal endonucleases. A structural comparison of SaCas9 with SpCas9 and AnCas9 revealed that the RuvC and HNH domains are connected by α-helical linkers, L1 and L2, and that notable differences exist in the relative arrangements between the two nuclease domains. A biochemical study suggested that PAM duplex binding to SpCas9 facilitates the cleavage of the target DNA strand by the HNH domain. However, in the PAM-containing quaternary complex structures of SaCas9 and SpCas9, the HNH domains are distant from the cleavage site of the target DNA strand. A structural comparison of SaCas9 with Thermus thermophilus RuvC in complex with a Holliday junction substrate indicated steric clashes between the L1 linker and the modeled non-target DNA strand, bound to the active site of the SaCas9 RuvC domain. These observations suggested that the binding of the non-target DNA strand to the RuvC domain may facilitate a conformational change of L1, thereby bringing the HNH domain to the scissile phosphate group in the target DNA strand.


Structure-Guided Engineering of SaCas9 Transcription Activators and Inducible Nucleases
'''Structure-Guided Engineering of SaCas9 Transcription Activators and Inducible Nucleases'''
Using the crystal structure of SaCas9, we sought to conduct
 
structure-guided engineering to further expand the CRISPRCas9
Using the crystal structure of SaCas9, we sought to conduct structure-guided engineering to further expand the CRISPR-Cas9
toolbox, as we have done previously using the SpCas9
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.
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 (Nishimasu
et al., 2014; Anders et al., 2014) (Figure S4D), and
permitted the insertion of RNA aptamers into the sgRNA to
create robust RNA-guided transcription activators (Konermann
et al., 2015). To generate the SaCas9-based activator system,
we created a catalytically inactive version of SaCas9 (dSaCas9)
by introducing the D10A and N580A mutations to inactivate the
RuvC and HNH domains, respectively, and attached VP64 to
the C terminus of dSaCas9 (Figures 7A and 7B). 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 (Figure 7A). To evaluate the dSaCas9-
based activator design, we constructed a transcriptional
activation reporter system, consisting of tandem sgRNA target sites upstream of a minimal CMV promoter driving the expression
of the fluorescent reporter gene mKate2 (Zhang et al.,
2011) (Figure 7B). We included an additional transcriptional
termination signal upstream of the reporter cassette, to reduce
the background previously observed in a similar reporter (Cong
et al., 2012) (Figure 7B). We observed robust activation of mKate2 transcription when we expressed the engineered sgRNA
complementary to the target sites, whereas the non-binding
sgRNA had no detectable effect (Figure 7C). Based on a
screening of different sgRNA designs with this reporter assay,
we 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
(Figure 7D). 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. We
selected two guides 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 (Konermann et al.,
2013) (Figure 7E). 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 (Zetsche et al., 2015; Wright et al., 2015), which
can be further engineered into an inducible system (Zetsche
et al., 2015). Our SaCas9 structure revealed several flexible regions
in SaCas9 that could likewise serve as potential split sites
(Figure 7F). We created three versions of a split-SaCas9, and two
of them showed robust cleavage activity at the endogenous
EMX1 target locus (Figure 7G). Using the best split design, we
then tested inducible schemes based on the abscisic acid
(ABA) sensing system (Liang et al., 2011), as well as two versions
of the rapamycin-inducible FKBP/FRB system (Banaszynski
et al., 2005) (Figures 7H and 7I). 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 (Figure 7J).


=See aslo=
=See aslo=

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Alexander Berchansky, Michal Harel
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