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Genetics / Genomics JoVE (Open Access) Citable · DOI

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

· 2015
DOI: 10.3791/52118
What you'll learn
  • ✓ Design and validate CRISPR/Cas9 guide RNAs for targeted genomic deletions
  • ✓ Transfect mammalian cells with CRISPR components and isolate edited populations
  • ✓ Screen and identify clonal cell lines with confirmed deletions via PCR and sequencing
  • ✓ Apply CRISPR/Cas9 methodology to disrupt genes in cultured mammalian cells
Protocol

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Difficulty
advanced
Total time
~2–3 weeks (transfection ~2 hrs, cell culture ~7–10 days, screening/cloning ~5–7 days)
Model organism
MEL cells (murine erythroleukemia)
Biosafety
BSL-1

Steps

1
Transfect CRISPR/Cas9 components into mammalian cells

Introduce CRISPR guide RNA and Cas9 protein/plasmid into MEL cells using standard transfection methods. Monitor transfection efficiency and cell viability post-transfection.

▶ 01:22
2
Sort transfected cells by fluorescence-activated cell sorting

Enrich successfully transfected cells using FACS based on fluorescent marker expression. Collect sorted populations for downstream screening and cloning.

▶ 02:35
3
Screen bulk cultures for CRISPR-mediated genomic deletions

Design and validate PCR primers spanning the target deletion region. Screen bulk transfected cell populations for presence of deletion alleles via PCR amplification and analysis.

▶ 03:41
4
Clone and characterize CRISPR-edited cell lines

Perform single-cell cloning from positive bulk cultures. Screen individual clones by PCR and DNA sequencing to confirm homozygous or heterozygous deletions.

▶ 05:26
5
Validate successful genomic deletion in clonal populations

Demonstrate Pim1 deletion in selected MEL clones using PCR product analysis and confirmation of expected molecular weight shifts relative to wild-type controls.

▶ 07:10
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