Conversion Tract Analysis of Homology-Directed Genome Editing Using Oligonucleotide Donors

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1999; p. 131
• Main Authors: Kan, Yinan, Hendrickson, Eric A
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Cell Line; CRISPR-Cas Systems - genetics; Gene Editing - methods; Genes, Reporter - genetics; Genetic Vectors - genetics; Humans; Lentivirus - genetics; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Oligonucleotides - genetics; Primary Cell Culture - methods; Recombinational DNA Repair; Transfection - methods; Homology-directed repair (HDR); Oligonucleotide; Conversion tract; Gene conversion; Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) {CRISPR-Cas9}; Gene editing
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9500-4_7

Homology-directed genome editing is the intentional alteration of an endogenous genetic locus using information from an exogenous homology donor. A conversion tract is defined as the amount of genetic information that is converted from the homology donor to a given strand of the targeted chromosomal locus. Because of this, conversion tract analysis retrospectively not only elucidates the mechanism of homology-directed genome editing but also provides valuable insights on the conversion efficiency of every nucleotide in the homology donor. Here we describe a blue fluorescent protein-to-green fluorescent protein conversion system that can be conveniently used to measure the efficiency and analyze the lengths of conversion tracts of homology-directed genome editing using oligonucleotide donors in mammalian cells.