pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

CRISPR/Cas9 Genome Editing
CRISPR/Cas9 reagents pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330, Addgene 42230) and its derivative pSpCas9(BB)-2A-GFP (pX458, Addgene 48138), were obtained from the Zhang laboratory via Addgene (33), as were the CRISPR design tools based on the mm9 genome assembly for a 20-nucleotide sequence followed by a protospacer adjacent motif (PAM) from Streptococcus pyogenes of NGG. All guides were selected for the least number of potential off-target sites and, where possible, the fewest potential off-target sites within coding exons. Guide sequences (sequence-PAM) were as follows (a “g” was added preceding the sequence as necessary for the U6 promoter): Mmp13-Pro (338-bp deletion), G1-gTTCTGCCACAAACCACACTT-AGG and G2-GCCTTCAAGGAAATACAGCA-AGG; Mmp13 −10k (427-bp deletion), G1-GGTCCTGGCCTTAGGTGAGC-GGG and G2-gCTGAGGCCAACTGGTTCAA-AGG; Mmp13 −30k (582-bp deletion), G1-gAGAAGCAACCTACCTACTCA-TGG and G2-GCTGTAGCCCTCGTGAGTCC-AGG; VDRKO (exon 3, 55-bp deletion): G1-gAGTGTGTGGAGACCGAGCCA-CGG and G2-gCGGTCAAAGTCACCAGGGTC-AGG; RUNX2KO (exon 3, 33-bp deletion), G1- gCTGTGGTTACCGTCATGGCC-GGG and G2-gCCCATCTGGTACCTCTCCGA-GGG. Guide sequences (without PAM) were cloned into pX330 or pX458 and co-transfected into UAMS-PB cells using FuGENE HD (Promega, Madison, WI) as per the manufacturer's recommendations scaled to a 10-cm plate. After 48 h, transfected cells were subjected to fluorescence-activated cell sorting (FACS) to acquire GFP-positive cells (130-μm tip and 15 p.s.i.) that were individually placed into single wells of a 96-well plate by the University of Wisconsin Madison Flow Cytometry Core. Isolated cell clones were grown to confluence and tested using genotyping primers (available upon request) that spanned the deleted genomic region, both junctions, and an internal set of primers. The VDR and RUNX2 KO clones were further tested by Western blot analysis. Clones were also PCR-amplified and sequenced to verify the deleted genomic material.

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Deletion 3T3-L1 MmP13 using pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Deletion 3T3-L1 MmP13 using pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.