96 well shuttle system

Cas9 RNP was prepared immediately before experiment by incubating with sgRNA at 1:1.2
molar ratio in 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM MgCl₂, 10% glycerol
and 1 mM TCEP at 37°C for 10 min. HDR template was then added to the RNP
mixture. Cells were dissociated by 0.05% trypsin, spun down by centrifugation at
400×g for 3 min, and washed once with DPBS. Nucleofection of
HEK293T cells was performed using Lonza (Allendale, NJ) SF cell- kits and program
CM130 in an Amaxa 96-well Shuttle system. The human neoFB were transfected with Lonza
P2 kit and program CA137. The hES cells were transfected with P3 primary cell kit and
program CB150. Each nucleofection reaction consisted of approximately 2 ×
10⁵ cells in 20 μl of nucleofection reagent and mixed with 10
μl of RNP:DNA. After electroporation, 100 μl of growth media was added to
the well to transfer the cells to tissue culture plates. The cells were incubated at
37°C for 24 hr, the media was removed by aspiration, and 100 μl of Quick
Extraction solution (Epicentre, Madison, WI) was added to lyse the cells and extract
the genomic DNA. The cell lysate was incubated at 65°C for 20 min and then
95°C for 20 min, and stored at −20°C. The concentration of genomic
DNA was determined by NanoDrop (Thermo Fisher Scientific, Waltham, MA).

BFP-HEK cells were detached by 0.05% trypsin or gentle dissociation reagent, spun down at 600 g for 3 min, and washed with PBS. BFP-K562 cells were collected and washed with PBS. Nucleofection was conducted using an Amaxa 96-well Shuttle system following the manufacturer’s protocol, using 10 µL of Cas9 RNP (Cas9 - 50 pmole, crRNA and TracrRNA - 60 pmole unless the amount is specified) (Richardson et al., 2016 (link); Lin et al., 2014 (link); Schumann et al., 2015 (link)). Nucleofection with Donor DNA had an additional 100 pmole of Donor DNA. After the nucleofection, 500 μL of growth media was added to the cells and they were incubated at 37°C in their tissue culture plates. The cell culture media was changed 16 hr after the nucleofection, and the cells were incubated until flow cytometry analysis was performed. The NHEJ frequency was quantified by determining the BFP negative population, and the HDR frequency was quantified by determining the GFP positive population. For the chemically modified gRNA activity tests, chemically modified crRNAs (30 pmole) were nucleofected with TracrRNA (30 pmole), and Cas9 (25 pmole) into BFP-HEK cells (10⁵ cells).

Cells (2×10⁵ cells after counting) were detached by Accutase and spun down at 600 ×3 g for 3 min and then washed with PBS. The Amaxa 96-well Shuttle system was used for electroporation following the manufacturer’s protocol. Cpf1 RNP or Cas9 RNP with or without DNA (Cpf1: 50 pmol; crRNA: 50 pmol, Cas9: 50 pmol, sgRNA: 50 pmol, with or without ssDNA/ssRNA: 50 pmol) were prepared in 10 µl of electroporation buffer. After the electroporation, the cells were incubated at 37 °C in tissue culture plates with 500 μl of culture media. The culture media were changed 16 h after the electroporation.

Basal ERSI in stable IgG expressing cell lines was determined by transient expression of the RXG reporter plasmid using 96 Well Shuttle System (Lonza Biologics, Walkersville, MD) followed by flow cytometry in high throughput format using an LSRII instrument (BD Biosciences, San Jose, CA). Basal ERSI is the measure of ER stress while the cells are in maintenance culture, and not in fed-batch culture. ERSI was calculated from the statistics generated by FlowJo software (TreeStar Corporation, Ashland, OR) using the formula described in the Results.

The lead gRNA (Mm. Cas9.APOE.1-E) resulted in a 99% INDEL frequency with no in-frame INDELs and was electroporated into B16-F10 cells using the Lonza 96-well Shuttle System as previously described. The electroporated cells were plated in 1 well of a 6-well plate and allowed to grow until confluent. The cells were then dissociated by trypsinization, resuspended in media, and counted. The suspension was diluted to 20,000 cells/mL; 4000 cells were added to 1 well of a 96-well plate and then diluted by array dilution. After 5 days of growth, each well was visually screened for single colonies. Wells with only 1 colony were allowed to grow to confluency. Each well was progressively passaged to a larger well until confluent in a 100 mm dish, about 8.8 x 10^6 cells for genomic DNA extraction and further cell passaging. The genomic DNA from each well was subject to quantification of total editing and analysis of INDEL profile by NGS to confirm a monoclonal isolate.