• 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

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Bacterially expressed Cas9 protein

In vitro transcribed/synthetic gRNA

Cas9 gRNA guided DNA nuclease RNP

RNP drug introduced into isolated cells

+