Human AMPKαcatalytic subunit encompasses two isoforms,
AMPKα1 and AMPKα2, encoded by thePRKAA1andPRKAA2
genes located at chromosomes 5p13.1 and 1p32.2, respectively.
PRKAA1is made of 9 exons (Fig. 1 A) representing 5088 base
pairs (bp) and encodes a 559-amino acid (AA) protein of 64 kDa.
PRKAA2coding sequence is 9280 bp long but with a long 3^0
untranslated region and encodes a 552-AA protein of 62 kDa
(Fig. 1 B). To targetPRKAA1gene, we designed two sgRNAs
targetingPRKAA1exon 7. To targetPRKAA2gene, we designed
two sgRNAs targetingPRKAA2exon 1 (Fig. 1 AandB).CRISPR Design- Identify sgRNA targeting sequences in the genomic region of
interest by running the CRISPR design tool (seeNotes 6– 8 ).
The output window shows 23 bp genomic sites of the form
50 -N 20 NGG-3^0 within your target region. These sites may
reside on theþorstrand (Fig.2). - Remove NGG sequence to leave 20 nt target sequence.
- Add G to the 5^0 end of target sequence if it does not begin with
G. (SeeNote 9). - For cloning into the plentiCRISPR vector using BbsI restric-
tion enzyme, add GATC to the 5^0 end of forward oligonucleo-
tide, and add AAAA to the reverse complement
oligonucleotide (including any additional G nucleotide). (See
Note 10). - Synthetize forward and reverse oligonucleotides
corresponding to the selected sgRNAs. (SeeNotes 11and 12 ).
CRISPR Cloning - Mix 1μl of 100μM of forward and reverse oligonucleotides
with 1 U of T4 PNK in 10μlof1kinase buffer, and incubate
for 30 min at 37C. (SeeNote 13). - Incubate at 95C for 5 min and gradually cool the solution at
room temperature. - Digest 5μg of plentiCRISPRv2 plasmid backbone with 30 U
of Esp3I (BsmBI) in a final volume of 60μlof1FastDigest
Green Buffer. Incubate for 25 min at 37C and then at 65C
for 15 min. (SeeNote 14). - Prepare a 10μl ligation reaction mix by adding 150 ng of
plentiCRISPR digestion product, 1 μlof1μM annealed
sgRNA oligonucleotides, 1μlof1U/μl T4 ligase, 1μlof
10 T4 ligase buffer. Incubate for 10–30 min at room
temperature.
176 Adrien Grenier et al.