AMPK Methods and Protocols

3. Many bioinformatics tools are available online to help in the
   design of sgRNA (e.g., ZiFit, CRISPR Design Tool). We used
   the optimized CRISPR Design application software from
   Dr. Feng Zhang’s laboratory (http://crispr.mit.edu). This soft-
   ware provides an accurate analysis of off-target sites. Due to the

AMPKa 1

β-actin

MOLM-14

AMPK#1_9AMPK#1_23AMPK#1_26AMPK#1_28AMPK#1_34AMPK#2_10AMPK#2_13AMPK#2_15AMPK#2_16AMPK#2_22AMPK#2_23AMPK#2_30

CTRAMPK#1_1AMPK#1_3AMPK#1_10AMPK#1_13AMPK#1_16AMPK#1_19AMPK#1_22AMPK#1_23AMPK#1 25AMPK#1_33AMPK#1_35AMPK#1_40CTRAMPK#1_42AMPK#1_50AMPK#2_3AMPK#2_5AMPK#2_6AMPK#2_8AMPK#2_10AMPK#2_12AMPK#2_14AMPK#2_22AMPK#2_25AMPK#2_29

AMPKa 1

β-actin

OCI-AML3

CTRAMPK#1AMPK#2CTRAMPK#1AMPK#2

MOLM-14 OCI-AML3

AMPKa 1

β-actin

A

C

B

Fig. 5AMPKα1 protein expression in sgRNA AMPKα1 AML cell lines. (A) Western blot analysis of AMPKα 1
protein expression in MOLM-14 and OCI-AML3 cell lines bulk populations transduced with lentivirus expres-
sing CRISPR/AMPKα1#1 and #2. (B) Western blots analysis of AMPKα1 protein expression in single-cell
OCI-AML3 clones after transduction with lentivirus expressing CRISPR/AMPKα1#1 and #2. (C) Western blots
analysis of AMPKα1 protein expression in single-cell MOLM-14 clones isolated after transduction with
lentivirus expressing CRISPR/AMPKα1#1 and #2. Clone numbers highlighted inredand inblackindicate
successful AMPKα1 knockout and knockout failure, respectively. Western blots were performed using anti-
AMPKα1 and anti-α-actin antibodies

188 Adrien Grenier et al.