Alt-R™ CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improved genome editing
Probing The Quality of Cas9 Ribonucleoprotein Complex ... · Differential Scanning Fluorimetry...
Transcript of Probing The Quality of Cas9 Ribonucleoprotein Complex ... · Differential Scanning Fluorimetry...
• Editas Medicine is developing CRISPR Cas9, an RNA-guided DNA nuclease, as a gene editing therapeutic. In ex vivo gene editing, isolatedcells will be modified by direct delivery of the Cas9-gRNA ribonucleoprotein complex (the Cas9 RNP) (Figure 1).
• This approach requires a detailed characterization of the Cas9 RNP complexwith regard to composition, stability, and potency among others.
• To assess the complexation quality of the RNP, differential scanning fluorimetry (DSF), a well-established biophysical assay, was used to measure the thermostability of RNPs (Figure 2).
• Two plate types were screened to optimize the DSF fluorescence signal fidelity. White coated plates otherwise optimal for fluorescence measurements showed spurious peaks whereas clear bottom plates showed only one peak indicating a clear melting transition which allows for accurate melting temperature (Tm) measurement (Figure 3). The higher the Tm the greater is the stability of the RNP complex.
• To characterize the RNP from two gRNA formats (full length and truncated) we examined the DSF profiles and biochemical cutting activity (Figure 4). Truncated gRNA (lengths < 80 nucleotides) which are easier to obtain synthetically show reduced efficacy and lower stability.
• Proper folding of this gRNA is likely required for optimal RNP complexation and nuclease activity. We investigated the effect of folded and misfolded or aggregated gRNA on RNP complexation by DSF (Figure 5). Misfolded gRNA destabilized the Cas9 protein reducing its Tm as compared to Apo Cas9. Finally, RNP complexes from misfolded gRNA were able to edit cells less efficiently than those from properly folded gRNA (Figure 6).
• These results indicate that gRNA format and folding influence RNP stability and activity. Understanding the impact of folding on efficient and inefficient gRNA will significantly help improve the fidelity of screening campaigns to identify the optimal gRNA to mediate a genomic edit.
Probing The Quality of Cas9 Ribonucleoprotein Complex
Using Biochemistry and Biophysics
1Northeastern University, 360 Huntington Ave, Boston, MA 02115, 2Editas Medicine, 11 Hurley St. Cambridge, MA 02141
Background
Differential Scanning Fluorimetry (DSF)
Conclusions
• We demonstrate the characterization of the Cas9 RNP complex by an optimized differential scanning fluorimetry assay.
• We show that the folding of the gRNA affects RNP complexation and has an impact on the activity of the RNP.
• The combined use of DSF, biochemical cutting and cellular potency will help to understand the various determinants of RNP stability towards enabling its use as a therapeutic.
DSF Can Distinguish Folded and Misfolded gRNA RNP Complexes
Misfolded
FoldedApo Cas9
Misfolded
Folded
0%
19%
38%
56%
75%
12.2 6.1 3.05 1.53 0.77 0.38 0.19
Cutt
ing
Dose (pmols)
Cellular Cutting Assay
Misfolded Folded
Apo Cas9
-2362.50
-1575.00
-787.50
0.00
787.50
1575.00
20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 71 74 77 80 83 86 89 92 95
RFU
Temperature (℃)
DSF Using White Coated Plates
A1 A4 A9 B3Buffer RNA Apo Cas9 RNP
-1500.00
-1125.00
-750.00
-375.00
0.00
375.00
20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 71 74 77 80 83 86 89 92 95
RFU
Temperature (℃)
DSF Using Clear Plates
A1 A2 A7 A13Buffer RNA Apo Cas9 RNP
Assay Optimization: Clear Bottom Plates Give Optimal DSF Signal
Figure 2: DSF works by monitoring the unfolding of a protein using sypro-orange dye. Unfolding of the protein
exposes hydrophobic regions causing dye to bind and be less quenched than in free solution resulting in an increase
in fluorescence (left). DSF profiles are analyzed as a second derivative of the fluorescence signal (right) where the
trough indicates the melting point (Tm) of the complex being studied.
Figure 5: DSF profile (left) and native PAGE gel (right) of folded and misfolded or aggregated gRNA.
Misfolded gRNA tend to destabilize the RNP complexes and show lower Tms.
Figure 6: Cellular cutting efficiency of misfolded (blue) and properly folded (orange) gRNA. A higher
amount of misfolded gRNA complex is required to get efficient cutting. The ability of improperly folded
gRNA RNP complex to cut in cells suggests a possible ensemble of folded/unfolded states resulting in a
reduced potency.
Guillaume Harmange1, Eric Tillotson2, William Selleck2, Hari Jayaram2
Spurious peaks
Truncations To gRNA Affect Biochemical Activity
0%
25%
50%
75%
100%
8:1 6:1 4:1 2:1
% S
ubst
rate
Cle
aved
Ratio RNP:DNA
In vitro Cutting Assay
Truncated Full Length
Apo Cas9
Truncated
Full Length
Figure 4: Comparison of in vitro biochemical cutting activity of truncated (blue) and full-length (orange)
gRNA (left). DSF profile of truncated and full-length gRNA (right). Truncated gRNA complexes are less
stable (lower Tm) and show less efficient biochemical cutting.
Ex vivo Gene Editing Workflow
RNP With Misfolded gRNA Cut Less Efficiently
Figure 3: Comparison of DSF signal obtained using white coated plates (left) with clear bottom plates
(right) across multiple CFX96 Real-Time PCR detection systems. The white coated plates irreproducible
showed spurious peaks at 60 °C as compared to clear bottom plates that did not show this across
multiple RT-PCR machines tested.
Figure 1: Purified Cas9 protein from bacteria are combined with in vitro transcribed or synthetic gRNA to form the
Cas9 RNP. This RNP complex is then introduced into cells isolated from the patient to carry out the genomic edit.
Edited cells are then introduced back into the patient.
RNP Apo Cas9
RNP
Apo Cas9
+ Deletion
Mutation
Example of Cut and Remove edit
saCas9
cgt 2016-04-27 12hr 40min 35bromoacetate 67thiol final shipped annotated edt free
Printed: 5/31/2016 12:45:36 PM Page 1 of 1Location: C:/Users/dcarlson/Desktop
Bacterially expressed Cas9 protein
In vitro transcribed/synthetic gRNA
Cas9 gRNA guided DNA nuclease RNP
RNP drug introduced into isolated cells
+