Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew?

Figures

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  Figure 1. Genome editing tools and delivery methods. (A) Classes of genome editing tools currently used in laboratory studies. Schematic adapted from ref (1). (B) Delivery methods for introducing CRISPR/Cas9 and other genome editing biomolecules into cells.

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  Figure 2. Genome editing tools and delivery methods. Inputs in the editing process. (A) Analysis results of 30 publications using CRISPR/Cas9 to edit HEK293 or HEK293T cells. Units reported in these publications are presented for gRNA (rows) and Cas9 (columns), with the number of publications that reported each combination shown in the matrix. (B) Pie chart of the number of publications in the analysis that used a given type of editing biomolecule format. (C) Pie chart of the number of publications that reported a given type of units for the cells used in the editing reaction, with N defined as the number or quantity of the cell population. (D) IVC assay using published commercial protocols54 showing the percent cleavage efficiency of two human gene targets EMX1 and HPRT1 with the indicated concentrations of the Cas9, gRNA, and template DNA. Error bars are means ± the standard deviation of two replicate reactions. (E) Key input quantities necessary for accurately reporting on and being able to reproduce an RNP editing reaction.

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  Figure 3. Verifying reagent delivery. (A) Example of three types of fluorescent reporters used to confirm delivery of the solution into cells: lucifer yellow (LY), tracrRNA conjugated to an ATTO fluorophore, and Cas9 fused to a fluorescent protein (FP) such as GFP or mCherry. (B) Possible evaluation methods by which fluorescent reporters for RNP delivery could be measured. (C) IVC assay with RNP formulated in a 1:1.4 Cas9:gRNA ratio, 366 nM Cas9, 500 nM two-part gRNA with tracrRNA-ATTO550, and 4.52 mM lucifer yellow (LY), testing the impact of fluorescent tracers on the cleavage efficiency of the DNA template EMX1.78 Error bars are means ± the standard deviation of two replicate reactions.

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  Figure 4. Single-cell manipulation and measurement. (A) A conceptual model of possible future advancement in genome editing experiments is to target specific cells in a population for delivery of the treatment condition and culture them alongside cells with the control condition, in the same environment. This could be done with direct microinjection or photostimulation with controlled arrays to direct the excitation wavelength to a specific area in a field of view. (B) Controlled delivery of a given quantity of reagent, and the ability to increment it intentionally, will allow for highly controlled dose response experiments. (C) Possible tools for relating the fluorescence intensity of a reporter to the volume of solution or number of editing molecules delivered to a cell.