CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Abstract

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Keywords: CRISPR-Cas; Cas9-CP; ProCas9; circular permutation; fusion proteins; genome editing; protein engineering.

Figure 1.. An unbiased Cas9 library screen identifies active circularly permuted Cas9 proteins.

(A) Overview of circular permutation and library generation for Cas9. (B) Enrichment values of the unbiased screen as determined by flow cytometry and colony forming units. Error bars indicate standard deviation in all panels. (C) Deep sequencing read averages for the Pre- and Post- Cas9-CP library members, demonstrating a strong clustering of highly enriched library members with internal (within 4 AA of the N- and C-termini) and empirically validated controls. The dotted line highlights an approximate boundary that represents >100-fold enrichment in the screen. (D) Model of new Cas9-CP termini (in red) based on PDB ID: 5F9R with domains colored according to the sequence bar (below). New termini are mapped onto the AA sequence bar. (E) Endpoint values for dCas9-CP 12 hr E. coli CRISPRi DNA binding and RFP repression system compared with WT dCas9 and a protein expression vector control in triplicate (error bars are s.d., * = p<0.05, ns = not significant, t-test). (F) CFU/mL readings in an E. coli genomic cleavage assay read out by cell death compared with a protein expression vector control, WT dCas9 and WT Cas9 (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test). (G) Cleavage efficiency of a genomic reporter in mammalian cells in triplicate (described in Figures S2B and S2C), observed via indel formation and GFP reporter disruption. hCas9 is human codon optimized Cas9; bCas9 indicates bacterial codon-based Cas9 constructs (error bars are s.d., * = p<0.05, ns = not significant, t-test).

Figure 2.. Linker length can be utilized to control Cas9-CP activity.

(A) Endpoint analysis of an E. coli CRISPRi based GFP repression assay run in triplicate using Cas9- CPs identified as functional with 20 AA linkers, evaluated with GGSⁿ linkers of length 5, 10, 15, 20, 25 and 30 AA. Error bars indicate standard deviation in all panels. (B) Schematic describing the rationale behind using a Cas9-CP with a short AA linker as a ‘caged’ molecule. (C) Endpoint analysis of an E. coli CRISPRi-based GFP expression time course with all six Cas9-CPs containing a 7 AA TEV linker (ENLYFQ/S) in the presence of a functional TEV protease (TEV, blue) compared with deactivated TEV protease with the catalytic triad mutant C151A (dTEV, gray) (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test). (D) Schematic and western blot against the Flag epitope on the C terminus of the CP-TEVs after the endpoint measurement (Figure 2C). Expected kDa indicates the predicted band size if cleavages occurs at the TEV site in the CP linker region.

Figure 3.. Generation of ProCas9s for sensing and responding to Potyvirus and Flavivirus proteases.

(A) Heat map depicting the fold activation of a suite of ProCas9 CP linkers for Potyviral NIa proteases. Data is normalized to a non-active protein expression control (dTEV) in an E. coli based CRISPRi GFP repression assay. Darker coloration indicates greater activity (n = 2). (B) Endpoint analysis of the E. coli CRISPRi assay utilizing the linker derived from Plum Pox Virus (PPV) comparing the response to distinct NIa proteases and a dead protease (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test compared to dProtease). (C) Heat map depicting the fold activation of a suite of ProCas9 CP linkers for Flavivirus NS2B-NS3 proteases, normalized to a non-active protein expression control (dTEV) in an E. coli based CRISPRi GFP repression assay. Darker coloration indicates greater activity (n = 2). (D) Endpoint analysis of the E. coli CRISPRi assay utilizing the linker derived from West Nile Virus (WNV) showing the response to distinct NS2B-NS3 proteases and a dead protease (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test compared to dProtease). (E) Schematic of the constructs used for the transient transfection and testing in HEK293T cells. (F) Mammalian GFP disruption assay (Figures S2A-C). HEK293T-based reporter cells were transfected with the indicated sgRNAs, WT Cas9 or a ProCas9 variant, and the respective proteases Reduction in GFP positive cells indicates genome cleavage by a Cas9 construct (n = 3, error bars are s.d., * = p<0.05, t-test compared to dProtease). (G) Flow cytometry plots from (F) with overlay of GFP-targeting (pink) vs. non-targeting (black) ProCas9^Flavi systems, demonstrating a small degree of background activity. (H) Truncation of ProCas9 AA linker sequence to prevent leakiness. (I) Leakiness and orthogonality of the original and shortened ProCas9^Flavi constructs. Displayed as percent GFP disrupted via normalization to the non-targeting guide for each construct-protease pairing. In addition to the deactivated protease (dProtease) control, the active Potyvirus NIa proteases were used to assess orthogonality (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test).

Figure 4.. ProCas9 stably integrated into mammalian genomes can sense and respond to Flavivirus proteases.

(A) Genomic integration and testing of Flavivirus protease-sensitive ProCas9s. HEK-RT1 genome editing reporter cells are stably transduced with various ProCas9 lentiviral vectors, followed by puromycin selection of ProCas9 cell lines. These cell lines are then either tested for leaky ProCas9 activity in the absence of a stimulus, or stably transduced with a vector expressing the indicated proteases, followed by assessment of genome editing using the GFP reporter. (B) Leakiness assessment of ProCas9 variants expressed from either the EFS or EF1a promoter. HEK-RT1 reporter cells were stably transduced with the indicated ProCas9 variants or Cas9-wt. Genome editing activity was quantified at the indicated days post-transduction. Error bars indicate the standard deviation of triplicates. (C) Leakiness assessment at the endogenous PCSK9 locus. HepG2 cells stably transduced with the indicated sgRNAs and ProCas9 variants or Cas9-wt. Cells were selected on puromycin and harvested at day 8 post-transduction for T7E1 analysis. (D) Mutational patterns and editing efficiency at the PCSK9 locus of samples shown in (C). Indels were quantified using TIDE. For clarity, the fraction of non-edited cells is represented as negative percentages. (E) ProCas9 leakiness quantification, as in (C), in A549 and HAP1 cells. Cells were selected on puromycin and harvested at day 7 post-transduction for T7E1 analysis. (F) Quantification of Flavivirus ProCas9 activation in response to various control (dTEV, pCF708) or Flavivirus (ZIKV, pCF709; WNV, pCF710) proteases. ProCas9 reporter cell lines were stably transduced with the indicated protease vectors. At day 3 post-transduction, cells were treated with doxycycline to induce GFP reporter expression. Error bars indicate the standard deviation of triplicates. Significance was assessed by comparing each sample to its respective dTEV control (unpaired, two-tailed t-test, n = 3, * = p<0.05, ns = not significant). (G) Genome editing activity in Flavivirus ProCas9 reporter cell lines, as in (F), at day 4 or 8 post-transduction. (H) Protease-sensitive editing at the endogenous PCSK9 locus. T7E1 assay of A549 and HAP1 Flavivirus ProCas9 cell lines (sgNT, sgPCSK9-4) stably transduced with the indicated mTagBFP2-tagged viral proteases. At day 4 post-transduction, mTagBFP2-positive cells were sorted and harvested for T7E1 analysis. (I) ProCas9^Flavi activation by Flavivirus (Flavi) proteases. *, small subunit of the activated ProCas9^Flavi (29 kDa). **, large subunit of the activated ProCas9^Flavi (137 kDa). (J) Immunoblotting for Cas9 in HEK293T co-transfected with plasmids expressing Cas9-wt or ProCas9^Flavi and dTEV or WNV proteases. The C-Cas9 (clone 10C11-A12) antibody recognize the large subunit of the activated ProCas9^Flavi (**, 137 kDa). The Flag-tag (clone M2) antibody recognizes the small subunit of the activated ProCas9^Flavi (*, 29 kDa). ***, likely small-subunit-ProCas9^Flavi-T2A-mCherry (55 kDa). Protein ladders indicate reference molecular weight markers.

Figure 5.. ProCas9 enables genomically encoded programmable response systems.

(A) CRISPR-Cas programmed cell depletion. HEK293T and HAP1 cells expressing Cas9-wt were transduced with mCherry-tagged sgRNAs. After mixing with parental cells, the fraction of mCherry-positive cells was quantified over time. Different sgRNAs targeting a neutral gene (sgOR2B6), an essential gene (sgRPA1), >100,000 genomic loci (sgCIDE) and a non-targeting control (sgNT) were compared. Error bars indicate the standard deviation of triplicates. (B) Competitive proliferation assay analogous to (A), conducted in HEK293T and HAP1 cells expressing the ProCas9^Flavi system. Note, sgCIDE positive cells show little or no depletion because the ProCas9^Flavi is in its inactive, vigilant state. (C) ProCas9^Flavi activation by Flavivirus proteases expressed from genomically integrated lentiviral vectors. (D) Competitive proliferation assay in HEK293T ProCas9^Flavi cells expressing the indicated mCherry-tagged sgRNAs, or a non-targeting control (sgNT) used for normalization. Cells were partially transduced with lentiviral vectors expressing a GFP-tagged dTEV or WNV protease, and cell depletion quantified by flow cytometry. Note, the WNV protease leads to protective cell death (altruistic defense) in sgCIDE expressing cells through activation of the ProCas9^Flavi system. Error bars indicate the standard deviation of triplicates. Significance was assessed by comparing each sample to its respective dTEV control (unpaired, two-tailed t-test, n = 3, * = p<0.05, ns = not significant).

Figure 6.. Application of Cas9 circular permutants.

Diagram showing various uses of Cas9 circular permutants (Cas9-CPs) as single-molecule sensor-effectors for protease tracing and molecular recording, or as optimized scaffolds for modular CP-fusion proteins with novel and enhanced functionalities.