Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage

Abstract

The CRISPR-Cas9 system from Streptococcus pyogenes has been exploited as a programmable RNA-guided DNA-targeting and DNA-editing platform. This evolutionary tool enables diverse genetic manipulations with unprecedented precision and ease. Cas9 is an allosteric enzyme, which is allosterically regulated in conformational activation, target recognition, and DNA cleavage. Here, we outline the underlying allosteric control over the Cas9 complex assembly and targeting specificity. We further review the strategies for mitigating intrinsic Cas9 off-target effects through allosteric modulations and the advances in engineering controllable Cas9 systems that are responsive to external allosteric signals. Future development of highly specific, tunable CRISPR-Cas9 systems through allosteric modulations would greatly benefit applications that require both conditional control and high precision.

Figure 1.

Allosteric regulation of CRISPR-Cas9. (a) Domain organization of Cas9. (b) Schematic representation of the Cas9 sgRNA base pairing with target DNA. The sgRNA functional modules are defined as in ref. [12]. (c) Diverse allosteric modulations leading to CRISPR-Cas9 with altered properties. Cas9 is subjected to allosteric regulation at different assembly stages following guide RNA loading and DNA binding, and its DNA targeting specificity and activity can be allosterically modulated through various RNA or protein engineering approaches. Allosteric modulators are shown in red.

Figure 2.

Stepwise conformational changes in Cas9 leading to cleavage activation. (a) The X-ray and cryo-EM structures of Cas9 reported from years 2014 to 2017 that highlight major conformational changes at each stage of assembly. Along the conformational activation pathway, these structures (shown clockwise) represent the apo state (PDB code: 4CMP), the sgRNA-bound pre-targeting state (PDB code: 4ZT0), the ssDNA-bound state (PDB code: 4OO8), the checkpoint state (PDB code: 4UN3), the pre-cleavage state (PDB code: 5F9R), and the pseudo-active state (PDB code: 5Y36), respectively. (b) The computational model and cryo-EM structures of Cas9 ternary complex presented in 2019 capturing the cleavage state (left), post-cleavage (middle; PDB code: 6O0Y), and product state (right; PDB code: 6O0X). The protein domains are color-coded as in Figure 1a, and the sgRNA, target DNA strand, non-target DNA strand are represented in orange, red, and green cartoon forms, respectively. Note that the split parts of REC1 are colored light and dark blue, respectively. For sgRNA, only the targeting region at its 5’ end is displayed for clarity (except 4ZT0). The alpha-carbon atoms of the active residues in the two nuclease domains (i.e. HNH and RuvC) are depicted as blue spheres. Major domain rearrangements of Cas9 are indicated with curved arrows. Note that the HNH and REC2 domains display alternate disorder transitions (highlighted with translucent ovals) in the post-cleavage and product complexes. Abbreviations: BH, bridge helix; PI, PAM-interacting domain; sgRNA, single guide RNA; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

Figure 3.

Allosteric modulation of Cas9 targeting accuracy through protein or sgRNA engineering. (a) Specificity enhanced Cas9 mutants obtained through the strategies of structure-guided rational design or directed evolution. The upper panel shows the timeline of various high-fidelity Cas9 variants reported to date. The PDB structure 5F9R is used for illustration of individual Cas9 domains where the mutations are located (left panel). The mutation positions for each Cas9 variant are marked in the right panel. eSpCas9, SpCas9-HF1, HypaCas9 and SpCas9^2Pro have been designed by rational approaches, and evoCas9, xCas9 3.7, and Sniper-Cas9 screened through directed evolution. (b) Schematic diagram of sgRNA modifications conferring improved specificity. Most of these modifications occur at the 5’ end of the sgRNA, including truncation of two or three nucleotides, extension of two guanine nucleotides, and partial DNA replacement. The incorporation of chemically modified nucleotides, such as 2’-O-methyl-3’-phosphonoacetate (MP) and bridged nucleic acids (2’,4’-BNA^NC[N-Me]) at specific locations to the 5’ end or within the central region of the sgRNA guide has also been demonstrated to broadly reduce off-target cleavage.

Figure 4.

Selected case studies demonstrating allosteric control over Cas9-guide RNA function through protein or guide RNA engineering (a,c) or through direct modulation by small molecule inhibitors (b). These engineered systems enable conditional regulation of genome editing and transcription. (a) Insertion of a ligand binding domain from human estrogen receptor to Cas9 or insertion of a light-oxygen-voltage photosensor domain to AcrIIA4 (an anti-CRIPSR protein inhibiting Cas9) leads to switchable Cas9 systems by small molecule ligands (e.g. 4-hydroxytamoxifen) or light. (b) Cas9-DNA binding disrupted by small molecule inhibitors (e.g. the newly identified BRD0539). (c) Extending sgRNA to include small molecule-responsive aptazymes or aptamers imparts allosteric switch in Cas9 by regulating the accessibility of the sgRNA guide region to target DNA. The aptazymes or aptamers are modified to append an antisense sequence that base pairs with the sgRNA guide region in the absence of small molecules. Addition of a cognate ligand triggers ribozyme self-cleavage or riboswitch conformational change, promoting the release of the blocking sequence (i.e. antisense strand) from the guide RNA. Abbreviations: LBD, ligand binding domain; LOV, light-oxygen-voltage; Acr, anti-CRIPSR protein.