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What are the methods that can be used to validate the successful delivery of CRISPR components?
Posted March 14, 2024

Answer

The two main techniques to validate the successful delivery of CRISPR components are fluorophore expression and antibiotic resistance. 

  1. For fluorophore expression, vectors expressing fluorescent proteins (e.g. GFP) or tagging ribonucleoproteins (RNPs) with a fluorophore ensure efficient delivery. The success can be confirmed via fluorescence microscopy or FACS, and delivery efficiency assessed by comparing the number of fluorescent cells to the total cell count. 
  2. Antibiotic resistancegenes (e.g. puromycin-N-acetyltransferase) in vectors help confirm successful delivery of CRISPR reagents by enabling the survival and selection of transfected cells. However, while these markers validate delivery and expression of encoded nucleases, they do not ensure accurate targeting of the desired sequence.