Journal: bioRxiv

Article Title: Multimodal control of Cas13 activity through domain insertion at an allosteric hotspot

doi: 10.1101/2025.04.21.649818

Figure Lengend Snippet: (a) Schematic depicting LightR and UniRapR domains inserted between residues QK634 in RfxCas13d. (b) RfxCas13d-QK634-LightR demonstrates light inducible knockdown EGFP. (c) RfxCas13d-QK634-UniRapR demonstrates chemical inducible knockdown of EGFP. For b-c : error bars shown mean + 95% confidence interval for single cells in flow cytometry experiment. Data shown is from one experiment representative of n=3 replicates. (d) and (e) Knockdown of EGFP where crRNA is transfected in and expression of effector proteins is under a doxycycline inducible promoter for wild type RfxCas13d and RfxCas13d-QK634-LightR, respectively. (f) Histogram showing robust EGFP knockdown (∼100-fold) of cells that stably express EGFP, RfxCas13d-LightR and crRNA targeting EGFP mRNA with light treatment.

Article Snippet: UniRapR sequence was cloned from pUSE-Src-YF-UniRapR-mCerulean-myc (Addgene #45381) 31 .

Techniques: Knockdown, Flow Cytometry, Transfection, Expressing, Stable Transfection

Journal: Nature Communications

Article Title: Two-input protein logic gate for computation in living cells

doi: 10.1038/s41467-021-26937-x

Figure Lengend Snippet: Chop -FAK is allosterically regulated by inserted sensor domains uniRapR and LOV2, which serve as input response elements. Rapamycin and light are the input signals for uniRapR and LOV2, respectively. From the sensor domains signals propagate through the amino acid core network (shown as contact network) of the protein. The output is FAK activation.

Article Snippet: We amplified the genes encoding uniRapR and LOV2 from Addgene vectors (Addgene plasmids #45381 and #87356, respectively).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Two-input protein logic gate for computation in living cells

doi: 10.1038/s41467-021-26937-x

Figure Lengend Snippet: a Ribbon diagrams of FAK from the published crystal structure (PDB ID: 2JOJ). Loop 1 is the insertion site for uniRapR (left), and Loop 2 is the insertion site for LOV2 (bottom). Green paths represent signal propagation pathways from the insertion sites to the target region (circled). b Contact map computed from the published crystal structure (PDB ID: 2JOJ) for sequence conservation and surface exposure. Loop 2 (L2) has low sequence conservation and high surface exposure. Bottom figure demonstrates the β-hairpin loop (loop 2) within the FERM domain. c Top, snapshots from the DMD simulations showing conformations of Op -lit-FAK and Op -dark-FAK. The distance between the two domains in Op -dark-FAK is indicative of activation. Bottom, frequencies of structures with given distances between FERM and kinase domains in Op -lit-FAK and Op -dark-FAK quantified during DMD simulations. Source data are provided as a Source Data file.

Article Snippet: We amplified the genes encoding uniRapR and LOV2 from Addgene vectors (Addgene plasmids #45381 and #87356, respectively).

Techniques: Sequencing, Activation Assay

Journal: Nature Communications

Article Title: Two-input protein logic gate for computation in living cells

doi: 10.1038/s41467-021-26937-x

Figure Lengend Snippet: a Schematic representation of the Op -FAK. LOV2 domain is inserted into the FERM domain of FAK. Light is used as input signal. Dark condition activates FAK and light conditions inactivates FAK. b Images of cells transfected with Op -dark-FAK (left) and Op -lit-FAK (right) on a fibronectin-coated glass surface. Inset shows the cell edges with arrows highlighting focal adhesions. Scale bar, 40 μm. c Normalized quantification of average size and total number of focal adhesions in Op -dark-FAK- and Op -lit-FAK-expressing FAK −/− cells on a fibronectin-coated glass surface. Data represent bar plots with mean±s.d. and individual data points, n = 10 cells for dark and lit mutants from 3 independent experiments; **** P = 6.2 × 10 −11 for number of focal adhesion (FAs), ** P = 0.0014 for size of FAs in mutants calculated by unpaired two-tailed Student’s t -test. NS, not significant. d Images of Op -lit-FAK- (left) and Op -dark-FAK-expressing (right) FAK −/− cells on PAA gels printed with fluorescently labeled fibronectin grids. Zoomed-in images show the well-formed focal adhesions along the grid lines for Op -dark-FAK. Scale bar, 40 μm; zoomed-in images, 14 μm. e Top, time-lapse images of FAK −/− fibroblasts that express Op -FAK exposed to light for the indicated periods of time. Bottom, magnification of region boxed in the upper left panel as a function of time in response to blue light (488 nm). Arrows indicate focal adhesions. Scale bar, 40 μm. f Normalized quantification of average size and total number of focal adhesions during light-induced inactivation of FAK −/− cells that express Op -FAK or Op -dark-FAK (control). FAs indicates focal adhesions. Data represent box plots and individual data points. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 10 cells for total FAs and average size of FAs from 3 independent experiments; *** P = 0.0003 for number of FAs, *** P = 0.0001 for size of FAs in Op -FAK. P = 0.7737 for number of FAs and P = 0.4617 for FAs size in control conditions calculated by unpaired two-tailed Student’s t -test. NS, not significant. Source data are provided as a Source Data file.

Article Snippet: We amplified the genes encoding uniRapR and LOV2 from Addgene vectors (Addgene plasmids #45381 and #87356, respectively).

Techniques: Transfection, Expressing, Two Tailed Test, Labeling, Control

Journal: Nature Communications

Article Title: Two-input protein logic gate for computation in living cells

doi: 10.1038/s41467-021-26937-x

Figure Lengend Snippet: a Left, schematic representation of ChOp -FAK. Validated Ch and Op modules were assembled to construct ChOp -FAK. Right, domain organization of ChOp -FAK. b Combinations of tested input conditions and the respective outputs. For Ch input, ChOp -FAK-expressing FAK −/− cells were treated with 50 nM rapamycin for 30 min. For Op input, dark and lit mutants of LOV2 were used to mimic the dark and light conditions, respectively. c Quantification of average size and total number of focal adhesions in FAK −/− cells expressing linker optimized ChOp -FAK. Pink box indicates inactivation and green box indicates activation of FAK. Data were normalized to Condition A. Focal adhesion (FAs) indicates focal adhesions. Data represent box plots and individual data points. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 18 cells for Condition A, n = 16 cells for Condition B, n = 15 cells for Conditions C and D from 3 independent experiments. For total number of FAs, **** P = 4.3 × 10 −12 for A and B, **** P = 1.07 × 10 −12 for A and C, **** P = 1.18 × 10 −13 for A and D, P = 0.08 for C and D. For Average size of FAs, **** P = 1.2 × 10 −16 for A and B, **** P = 7.25 × 10 −23 for A and C, **** P = 5 × 10 −21 for A and D, P = 0.768 for C and D conditions calculated by unpaired two-tailed Student’s t -test. NS, not significant. Source data are provided as a Source Data file.

Article Snippet: We amplified the genes encoding uniRapR and LOV2 from Addgene vectors (Addgene plasmids #45381 and #87356, respectively).

Techniques: Construct, Expressing, Activation Assay, Two Tailed Test

Journal: Nature protocols

Article Title: Engineering proteins for allosteric control by light or ligands

doi: 10.1038/s41596-019-0165-3

Figure Lengend Snippet: a) In rapamycin-regulated control (RapR), an insertable FKBP domain (iFKBP) is inserted at a surface loop where it allosterically inhibits the activity of the protein. Heterodimerization of iFKBP with co-expressed FRB is induced by addition of rapamycin (pink circle) to the medium, leading to activation of the target protein and productive interaction with downstream targets (P). Typically, constitutively active proteins are used so that activity is solely controlled by the user. b) The single chain/unimolecular version of this approach, called uniRapR, does not require co-expression of FRB, as the inserted domain (U) is a fusion of iFKBP and FRB. c) RapR-TAP enables allosteric activation of the target protein in a specified subcellular region, or when it is interacting specifically with one downstream target. Here iFKBP is inserted in the target, and the FRB required for activation is fused to the specific target protein, or to a subcellular targeting sequence. d) In protein photo-activation (PA), the LOV2 domain (L) is fused to the target protein, where it sterically blocks the active site. Blue light induces a LOV2 conformational change that reversibly uncovers the active site. e) In protein photo-inhibition (PI), LOV2 is inserted at a site where it allosterically perturbs the active site only in the light. f) The close proximity of the N and C termini of the iFKBP, uniRapR and LOV2 domains enables successful insertion into surface loops and subsequent allosteric regulation.

Article Snippet: REAGENTS Molecular cloning DNA encoding residues 1–198 of the uniRapR domain (Addgene Plasmid #45381) This plasmid is used to design single-chain rapamycin-induced allosteric proteins.

Techniques: Control, Activity Assay, Activation Assay, Expressing, Sequencing, Inhibition

Journal: Nature protocols

Article Title: Engineering proteins for allosteric control by light or ligands

doi: 10.1038/s41596-019-0165-3

Figure Lengend Snippet: (A) The control with uniRapR should provide full activity with rapamycin and no activity without rapamycin. Conversely, in photo-inhibitable (PI) constructs, the protein should be deactivated with light. UniRapR-Src is deactivated without rapamycin, and the activity is rescued with rapamycin. Wild type or constitutively active (Src Y527F or YF) proteins should be used as positive controls, whereas catalytically inactive (kinase dead or KD) mutants can be used as negative controls. The expression of the constructs should be compared. Paxilin and focal adhesion kinase (FAK) are Src substrates. An alternative method should be also used to test the engineered constructs. In this case, the motility of mouse embryonic fibroblasts (MEFs) and translocation of PI-Src to focal adhesions are monitored upon photo-inhibition of PI-Src. (B) If significant activity is observed in the off state of a uniRapR-protein or in a PI-protein upon light treatment, the linkers between the sensory and host domain should be shortened. If insufficient activity is observed in the on state of uniRapR-protein or in the dark state of PI-protein, the linkers between the sensory and host domain should be elongated, or another insertion site should be tested. The same strategy should be pursued if there is lower expression of the constructs compared to controls. Some level of leakiness can be tolerated, depending on the biology and expression levels of the endogenous proteins. UniRapR-PAK1 insertion site is optimized by inserting uniRapR in between different residues of the loop. In another case uniRapR-PAK1 was generated by inserting uniRapR domain into a different loop (uniRapR-PAK1-L2). These figures were taken from published work1, 11, 13.

Article Snippet: REAGENTS Molecular cloning DNA encoding residues 1–198 of the uniRapR domain (Addgene Plasmid #45381) This plasmid is used to design single-chain rapamycin-induced allosteric proteins.

Techniques: Control, Activity Assay, Construct, Expressing, Translocation Assay, Inhibition, Generated

Journal: Nature protocols

Article Title: Engineering proteins for allosteric control by light or ligands

doi: 10.1038/s41596-019-0165-3

Figure Lengend Snippet: ?TROUBLESHOOTING

Article Snippet: To image transiently transfected HeLa cells, uniRapR-Src (Addgene, #45381) with a cell membrane marker (Addgene, # 54491) can be used.

Techniques: Activity Assay, Construct, Expressing, Sequencing, Plasmid Preparation, Transfection, Imaging