Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

Abstract

The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

“…Overlays of Cas9/sgRNA-DNA and Cas9/sgRNA structures reveal that in the Cas9/sgRNA structure, domains 2 and 3 of the helical recognition lobe sterically occlude the central channel in which the RNA/DNA hybrid is located in the Cas9/sgRNA-DNA complexes (16,21,26). This finding indicates that a structural rearrangement within the helical recognition lobe must occur upon target DNA binding and R-loop formation to avoid a steric clash between target DNA strand and the helical recognition lobe (21,29). We hypothesize that during the initial DNA interrogation steps preceding the DNA/sgRNA pairing, the helical recognition lobe maintains a conformation similar to the conformation seen in the Cas9/sgRNA complex (21), which should impede double-stranded DNA access to the central channel.…”

New CRISPR–Cas systems from uncultivated microbes

“…coli transformation assays for ARMAN-1 Cas9 and ARMAN-4 Cas9 were conducted as previously published 66 . Briefly, E. coli transformed with plasmids expressing guide RNA sequences were made electrocompetent.…”

Towards combinatorial transcriptional engineering

“…Modular approaches like the ones mentioned above are being explored for Cas9 also. Oakes et al, (2016) identified regions in Cas9 that are capable of tolerating insertions without affecting its function significantly. With this knowledge, they designed an allosterically regulated dCas9, which had an estrogen receptor ligand binding domain.…”

“…(Farzadfard et al, 2013;Jusiak et al, 2016;Konermann et al, 2015;PerezPinera et al, 2013;Tanenbaum et al, 2014;Zalatan et al, 2015) Can cause repression by steric hindrance, without the need for repressor fusions (Kabadi and Gersbach, 2014) Specificity of CRISPR/dCas9 based systems is higher than ZNFs, TALEs, Cas9 nucleases. Konermann et al, 2015) Orthogonal control of various genes is possible (Konermann et al, 2015;Yang et al, 2015;Zalatan et al, 2015) Conferring inducibilty to CRISPR/ Cas9 based systems is challenging (Jusiak et al, 2016;Kiani et al, 2014;Nihongaki et al, 2015;Nissim et al, 2014;Oakes et al, 2016;Shechner et al, 2015) Cas9 orthologs allow orthogonal control in synthetic circuits (Esvelt et al, 2013) Dynamic in nature due to more rapid RNA turnover rate (Kabadi and Gersbach, 2014) …”