Computational protocol: Identification and validation of regulatory SNPs that modulate transcription factor chromatin binding and gene expression in prostate cancer

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Protocol publication

[…] CRISPR/Cas9 target sites were identified using the E-CRISPR design tool available at, and targets were chosen with the best specificity. To create genomic deletions across the enhancer region of approximately 177–193-bp, two guides RNA were designed to the target sequences Guide RNAs were cloned into BbsI sites of pX458 vector (Addgene plasmid 48138), which encodes both the guide RNA, mammalian Cas9 enzyme with 2A-EGFP. 2 μg total px458 vector (encoding 1 μg each of guides#1 and guide#2) was introduced into LNCaP cells by Neon electroporation system (Life Technologies). Cells were resuspended in 500 uL of RPMI/10% FBS and incubated at 37°C with 5% CO2 for 48 hours. BIO-RAD S3™ Cell Sorter was used to capture the cells having high green fluorescent protein signals and then colonies were grown from single cells. Complete deletion of all alleles for a target locus was confirmed by PCR using primers flanking the enhancer. RT-qPCR analysis was performed in triplicate, comparing the deleted cells to parental LNCaP cells. […]

Pipeline specifications

Software tools CRISPR design, E-CRISP
Databases Addgene
Application Genome edition
Organisms Homo sapiens
Diseases Neoplasms, Prostatic Neoplasms