Journal: Drug Metabolism and Disposition

Article Title: CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism

doi: 10.1124/dmd.117.076307

Figure Lengend Snippet: CYP3A5 *3 allele (rs776746) and guide RNA targeting strategy. Guide RNAs (gRNAs) were targeted to PAM sequences on each side of the CYP3A5 *3 allele (gRNA1 or gRNA2 locus). Exon 3B sequence is in capital letters, and the upstream intron sequence is in lower-case letters. There are 77 bp between the gRNA guided Cas9 cut sites.

Article Snippet: A plasmid that expressed a human codon-optimized Cas9 ( Mali et al., 2013a – c ) nuclease was purchased from Addgene (Cambridge, MA) guide RNAs (gRNAs) targeting the CYP3A5*3 locus were designed using the CRISPR design tool ( http://crispr.mit.edu/ ).

Techniques: Sequencing

Journal: Drug Metabolism and Disposition

Article Title: CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism

doi: 10.1124/dmd.117.076307

Figure Lengend Snippet: Workflow for development of CYP3A5 genetically modified cell lines using CRISPR/Cas9 and clonal selection. The CYP3A5 *3 splice junction was deleted with gRNA1, gRNA2, and Cas9, or the CYP3A5 *3 SNP was point mutated using gRNA2 and an HDR template to convert the *3 guanine to a *1 adenine. After transfection, the cells were single-cell cloned by plating in soft agar. The single-cell clones were transferred to collagen I–coated plates until confluent. Cells were then expanded or screened by PCR of the CYP3A5 *3 locus and sequencing of the PCR products.

Article Snippet: A plasmid that expressed a human codon-optimized Cas9 ( Mali et al., 2013a – c ) nuclease was purchased from Addgene (Cambridge, MA) guide RNAs (gRNAs) targeting the CYP3A5*3 locus were designed using the CRISPR design tool ( http://crispr.mit.edu/ ).

Techniques: Genetically Modified, CRISPR, Selection, Transfection, Clone Assay, Sequencing