p3 primary cell nucleofection solution  (Lonza)

Journal: bioRxiv

Article Title: iSCORE-PD: an isogenic stem cell collection to research Parkinson’s Disease

doi: 10.1101/2024.02.12.579917

Figure Lengend Snippet: Schematic illustrating genome editing pipeline A. This approach involves (i) FACS-based enrichment of nucleofected cells containing the gene editing reagents including a fluorescent reporter, (ii) the isolation of clonally expanded cell lines and (iii) the NGS-based genotyping to identify correctly edited cell lines. Schematic illustrating genome editing pipeline B. This approach utilizes a high-throughput cell isolation system. This approach includes (i) nucleofection of the gene editing reagents, (ii) the plating of cells in a limited dilution (∼10 cells/well) to isolate wells containing correctly targeted cells by NGS and (iii) subcloning, expansion and NGS-based genotyping to isolate correctly targeted clonal cell line. Table summarizing the gene editing strategies used to generate the iSCORE-PD collection. These include: (i) CRISPR/Cas9 facilitated homology directed repair (HDR) using ssODNs containing the desired genetic modification as repair template for CRISPR/Cas9 induced double strand break. (ii) The use of competing HDR templates (ssODNs) containing synonymous mutations in the gRNA-target site to favor the generation of heterozygous over homozygous mutations. (iii) TALEN-facilitated HDR using ssODNs containing the desired genetic modification as repair template for CRISPR/Cas9 induced double strand break. (iv) Prime editing approach to insert the PD-associated point mutations into hESCs. (v) Dual CRISPR approach using 3’ and 5’ sgRNAs flanking the desired deletion to recreated large genomic structural alterations identified in PD patients. Overview depicting genome engineering and quality control steps in the generation of the iSCORE-PD collection.

Article Snippet: 5 x 10 5 to 1 x 10 6 cells were resuspended in 20µL of nucleofection solution (P3 Primary Cell 4D-Nucleofector™; Lonza) and nucleofected (Lonza 4D nucleofector TM Core + X Unit, program CA-137) using the following genome editing reagents for the corresponding edits described in : (1) Plasmid based CRISPR-Cas9 facilitated HDR: 200 ng gRNA plasmids (px330-GFP), 700 ng ssODN. (2) Plasmid based dual CRISPR: 500 ng 3’-gRNA plasmid (px330-GFP) and 500 ng 5’-gRNA plasmid (px330-mCherry). (3) TALEN facilitated HDR: 100 ng LRRK2-TALEN-TA01L and 100 ng LRRK2-TALEN-TA03R, 700 ng ssODN, 100 ng pEGFP-N1 (Clontech). (4) Plasmid based prime editing: 500 ng pCMV-PE2-GFP (a gift from David Liu, Addgene#132776) , 330 ng pU6-pegRNA and 170 ng pBPK1520-ngRNA. (5) RNP-based CRISPR-Cas9 facilitated HDR: 80 pmol purified Cas9 protein (QB3 Macrolab, UC Berkely), 300 pmol chemically modified synthetic sgRNA (Synthego) and 100 pmol ssODN HDR template. (6) RNP-based dual CRISPR: 80 pmol purified Cas9 protein, 150 pmol of each chemically modified synthetic 3’-sgRNA and 5’-sgRNA. (7) RNP-based CRISPR-Cas9 facilitated HDR with competing templates: 80 pmol purified Cas9 protein, 300 pmol chemically modified synthetic sgRNAs, 50 pmol ssODN HDR template carrying PD mutation and 50 pmol ssODN HDR template carrying a synonymous mutation. (8) RNA-based prime editing: 4 μg in vitro transcribed nCas9-RT mRNA, 100 pmol chemically modified synthetic pegRNA (IDT or Synthego) and 50 pmol chemically modified synthetic ngRNA (Synthego).

Techniques: Isolation, High Throughput Screening Assay, Cell Isolation, Subcloning, CRISPR, Modification