Site Directed Mutagenesis (SDM) Human Deletion SKOV3

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion 3T3-L1 MmP13

Get tips on using pSPCas9(BB)-2A-GFP plasmids to perform CRISPR Mouse - Deletion 3T3-L1 Usp2

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion ES (embryonic stem) cells Slx2

Get tips on using pSpCas9(BB)-2A-GFP (PX458) to perform CRISPR Mouse - Deletion ES (embryonic stem) cells MIR

Get tips on using pSpCas9(BB)-2A-Puro (PX459) V2.0 to perform CRISPR Mouse - Deletion ATDC5 MEK1

Get tips on using pSpCas9(BB)-2A-Puro (PX459) to perform CRISPR Mouse - Deletion ES (embryonic stem) cells Etv2 promoter

Get tips on using pSpCas9(BB)-2A-Puro (PX459) V2.0 to perform CRISPR Mouse - Deletion 3T3-L1 SWELL1

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Rat - Deletion INS-1 832/13 Ep300

Get tips on using MYH9 CRISPR/Cas9 KO Plasmid (h) to perform CRISPR Rat - Deletion PC12 myosin IIA (Myh9)

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 has 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 the efficacy of transduction and shRNA on its target site.