CRISPR Mouse Deletion L929

Get tips on using pHR-SFFV-KRAB-dCas9-P2A-mCherry to perform CRISPR Human - Repression GATA1

Get tips on using Phusion Site-Directed Mutagenesis Kit to perform Site Directed Mutagenesis (SDM) Human - Deletion MDA-MB-231 sodium channel β1 subunit

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion PC-3 AGR3

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion PC-3 AGR2

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion MCF-7 AHR

Get tips on using pAC154-dual-dCas9VP160-sgExpression to perform CRISPR Human - Activation HIV-1 5′ LTR

Get tips on using pSpCas9(BB)-2A-GFP (PX458) to perform CRISPR Human - Repression HPV-18 E6

Get tips on using pSpCas9(BB)-2A-GFP (PX458) to perform CRISPR Human - Repression HPV-18 E7

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion K562 c-Myb gene

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.