Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system is a promising tool for gene editing which allows efficient and directed alterations of any specific nucleic acids sequence in a genome. The guide RNAs (gRNAs) in these CRISPR systems are designed complementary to the target DNA sequences, which then direct the associated Cas9 protein to cause cleavage. However, the editing efficiencies of these gRNAs vary and depend on many factors such as their Guanine-Cytosine (GC) content, accessibility of target sites, etc. High-throughput screening of the gRNA libraries in cell culture media gives an estimate of their cleavage activity (both at on-targets and off-targets). Performing these high throughput screens inside mammalian cell lines is a cumbersome process due to the low efficiency of the transfection process. Moreover, we cannot perform these CRISPR screening assays,
in vitro, in just a bulk aqueous phase. There exist interactions between the different DNA templates in the oligo library and the individual gRNAs and their respective templates are not physically isolated. Water-oil emulsion technique enables the encapsulation of single DNA molecules in approximately, 10
8–10
9 micron-sized compartments of water droplets per ml of emulsion mixture. In these water-oil emulsions, if the reaction mixture is added to the aqueous phase, the non-interacting water droplets will serve as microreactors to perform the
in vitro CRISPR reactions in a high-throughput manner while simultaneously avoiding the effects of templates mispairing. However, according to the standard protocol for
In vitro digestion with Cas9 nuclease protein, the gRNAs specific to the target sites are first synthesized and purified. These purified gRNAs are then added to the reaction mixture to perform the CRISPR reaction. In this work, we have demonstrated how these CRISPR cleavage reactions can be performed in one pot, thereby skipping the external gRNAs synthesis step altogether. We further have demonstrated the advantage of carrying out the one-pot CRISPR
in vitro reaction in water-oil emulsion compared to the non-emulsified aqueous phase and its efficient use in high throughput CRISPR screening assays.
In this work, the emulsion-based system was prepared by bulk homogenization. A stable water-oil emulsion was generated with the mean water droplets size of 2 µm as confirmed by Dynamic Light Scattering measurements and Optical Microscopy. We first investigated the effect of change in the concentration of DNA templates in the non-emulsified phase. The result shows that the mispairing between the templates in the oligo library is prominent, even at a lower concentration. The reaction/cleaved products formed in the water-oil emulsion were compared to that in the non-emulsified phase. In the water-oil emulsion, a significant amount, ~ 40.3 % of the product, was formed from DNA templates isolated inside the water droplets, compared to almost none in the non-emulsified phase. Due to the presence of heterogenous droplets, templates mispairing was still evident in the water-oil emulsion, as observed in about 41% of the product. Further, a detailed comparative study will be done with water-oil emulsions generated using a droplet generator microfluidic system where uniform water droplets will be produced. Using microfluidics emulsification, we expect to eliminate the mispairing between the different templates.
