Reporter Strategy for CRISPR HDR Knock In

CRISPR HDR Knock In Strategy

1. Rapid Reporter Knock-in Summer 2015

2. Purpose: To develop a methodology to rapidly create cells in which a targeted gene has a fusion of a reporter or tag to facilitate the detection of endogenous RNA and protein levels

3. Abstract Inherited risk of complex disease is mediated through changes in functions of genes important in the disease pathology. The vast majority of genes and pathways that underlie complex disease risk is not known. Modern human population genetics have identified over 2600 differences between the genomes of individuals that correlate with inherited risk for many diseases such as cancer, heart disease, and type 2 diabetes. At present we do not know how these genomic differences termed (genetic variants) and the genes they affect (disease-genes), lead to changes in disease risk. The work described here is a methodology to rapidly measure the levels and localization of any disease-gene in living human cell models. Specifically we have developed a procedure to introduce a naturally fluorescent protein called green fluorescent protein (GFP) onto the ends of any target disease-gene creating a fusion between the disease gene and GFP (GFP-tag). To accomplish this we utilized modern genome editing tools (CRISPR/Cas9) to deliver a nuclease to locations at candidate disease genes. The Cas9 nuclease catalyzes precise changes of the genome necessary to integrate the GFP gene onto the end of the disease gene. Cells that have the gene-GFP tag are isolated and can be used for further experimentation. By measuring GFP as an easily detected readout of disease-gene levels high-throughput interrogation of disease variants that impact this gene can be identified in high-throughput screens of these variants. This has the potential to greatly expand our understanding of the molecular mechanisms that underlie inherited disease risk. Lastly, given a GFP-fusion of a candidate therapeutic target, this technology could be instrumental for testing potential therapeutics for impact of the levels of the gene .

4. Strategy 1 Exon reporter Isolate and genotype reporter-fusion Exon Cterm reporter Exon Exon Cterm Reporter targeting construct Homologous Recombination Target gene Readout target gene RNA/Protein: (fluorescence, IF, cell sorting, IP etc.) Exon Cterm reporter Exon Select cells by increase in reporter expression Synthetic transcription factor Induce target gene expression Exon Cterm

5. Strategy 2: Splicing dependent Knockin Exon Reporter Nterm Isolate and genotype reporter-fusion Exon Cterm Exon Exon Cterm Reporter targeting construct Homologous Recombination Target gene Readout target gene RNA/Protein: (fluorescence, IF, cell sorting, IP etc.) Exon Cterm Exon Reconstitute reporter by splicing Exon Cterm Reporter C term Reporter targeting construct Reporter Nterm Reporter C term N C Reporter Nterm Reporter C term

6. Time-line: 1-2 weeks – Design and reagent ordering 1-3 weeks – reagent construction Indefinite – pilot testing of each strategy Reagents: sgRNA for each target gene Cterm sgRNA for each target gene promoter Generalized Homologous recombination cassette ssODN for tag knock-in Targets: SLC16A11 TCF7L2 SLC16A13 HNF1B/A CDKN2A/B HNF4A IGF2BP2 IGF2

7. Steps of strategy 1 (part 1) Targeting construct design The location between the stop codon and the second to last codon was identified for every gene from a list of genes relevant in Diabetes The 80 base pairs before and after the stop codon in the 5’ and 3' direction, and their coordinates on the genome for every gene were identified. The 5’ 80 base pairs were then connected with the V5 Tag which was connected to the 3’ 80 base pairs, in the correct direction of the gene CRISPR/Cas9 design Small guide RNA binding sites were identified in the 5’ and 3’ directions of the 160 base pair complex Genotyping design A primer forward and primer reverse were then found in the 5’ and 3’ direction of every gene

8. Green Fluorescence Transfected positive Control cells Green Fluorescence protein treated cells Readout of fluorescence tagged gene No Green Fluorescence Control cells

9. Steps of procedure 1 (part 2) CRISPR construction These modified strands of DNA were ordered to be synthesized and were annealed and phosphorylated These modified oligonucleotides were then put into a vector of Crispr Cas9 The Crispr cas 9 was then put in bacteria cells and then divided into petri dishes and put into an incubator for expansion Vectors are isolated from bacteria by DNA purification