CAG-Cas9

CRISPR Mouse - Deletion Neuro 2a Rab38

Experiment
CRISPR Mouse - Deletion Neuro 2a Rab38
Product
CAG-Cas9 from Addgene
Manufacturer
Addgene

Protocol tips

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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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

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Papers

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Manufacturer protocol

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

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Videos

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