CAG-Cas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

4.2. CRISPR/Cas target sites and vector construction
CRISPR target sites were identified using http://crispr.mit.edu/ as described [34]. In order to create differently sized genomic deletions in Neuro2A cells, four different sgRNAs target sequences of 20 bp located upstream of a NGG PAM-sequence were selected. Selected target sequences (underlined) were cloned as complementary oligonucleotides in between two BbsI sites of a Bluescript plasmid containing a U6 promoter (pBS-U6) and the sgRNA backbone [34] for the production of RNA from the template sequence of sgRab38-2 cloned into pBS-U6-sgRab38-2: AAAACTTCTCCTCGCACTACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT, of sgRab38-3 cloned into pBS-U6-sgRab38-3:

GGCCCTCTCATCAAGAGCGACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT, of sgRab38-10 cloned into pBS-U6-sgRab38-10:

GTTTGTAAATATCCGCATATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT, of sgRab38-32 cloned into pBS-U6-sgRab38-32:

CTTGTGTAGCCGCACCCATGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT, and of sgRab38-52 cloned into pBS-U6-sgRab38-52:

TGCTTTTCCCAATAATCGTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

For the production of sgRNAs by in vitro transcription a T7 promoter sequence (TAATAATACGACTCACTATAG) was added upstream of the target sequence. To assess potential CRISPR off-target activity, three high-scored off-target sites (see Supplementary Fig. 2) were amplified by the locus-specific primer pairs shown in Table 2. For analysis the PCR products were purified, sequenced (GATC Biotech, Konstanz, Germany) and compared to wild type using the Vector NTI Advance 11.5 (Life Technologies) and Chromas (Technelysium) software. For the expression of Cas9 we replaced the TAL sequences of our pTALEN-pA plasmid with a T7 promoter and a codon optimized Cas9 coding region [35] purchased from Genscript (Piscataway, USA) to derive the pCAG-Cas9-pA expression vector. For the production of Cas9 mRNA we inserted the Cas9 coding region into our pT7-95A plasmid that was further modified by the addition of 67 A residues to derive the plasmid pCAG-Cas9-162A.

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Manufacturer protocol

Download the product protocol from Addgene for CAG-Cas9 below.

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