Genome-wide allele screen and chromosome knockout project
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Library Design Protocol:
- Input data is either phased haplotypes (in HapCut output format: File:Haplotype.chr21.combine.txt) or SNP Calls in tab delimited format (Snp Calls, Common European SNPs).
- Filter SNP calls or Haploytping data for quality
- Filter SNPs for snps that fall in PAM motifs (GG or CC) motifs
- Construct 23 bp spacer sequences + PAM motifs from these SNPs
- Using bowtie, check spacer sequences for multi-mapping. Ensure each spacer + PAM sequence is 2 or 3 mismatches away from any other location on the genome.
- Barcode each individual library from the bc_25mer set and order on gChip
- Run CRISPR Library Prep Protocol to prepare the libraries for incorporation
Genome-wide Allele Screen Plan:
- Sequence E.Coli cells to determine baseline library incorporation.
- Using lentiviral vectors, incorporate PGP1 specific and Common Allele libraries into PGP1 iPSCs.
- Differentiate for 2-3 weeks into embryoid bodies
- Using flow cytometer, flow-sort cells into 3 germ layers (endoderm, ectoderm, mesoderm)
- Look for enrichment/dropout over baseline.
Chromosome Knockout Plan:
- Use PGP1 iPSC cell line with mCherry reporter knocked in at BACH1 on Chr21 (only one of the two chromosomes. We will sequence SNPs near BACH1 to determine which chromosome has the reporter construct) and at SRY on ChrY. Successful deletion of the target chromosome should remove any mCherry activity.
- 05/05/2015 Library Order includes both full chr21 haplotype libraries and the chrY repeat library with scaffolds allowing for cloning in of a barcode. We can hybridize RNA-FISH probes to this barcode.
- Fix PGP1 iPSC cells (how?)
- Make pores in cells (how?)
- Using in-vitro translation kit, express nuclease-dead Cas9 and add our gRNA library.
- Hybridize gRNA-Cas9 to desired chromosome
- Hybridize RNA-FISH probes to gRNA scaffolds
- Wash cells to remove background
- Single-cell imaging