CRISPR/Cas9
plasmids were purchased from the Addgene repository. (Addgene plasmids
#78535 and 78547).38 (link) Two sgRNAs were selected
to target eGFP sequence (sgRNA1: GAGCTGGACGGCGACGTAAACGG;
sgRNA2: CAGAACACCCCCATCGGCGACGG). The
amino lipid carriers were dissolved in ethanol at a stock concentration
of 2.5 mM, while the plasmids of CRISPR/Cas9 system were reconstituted
in nuclease-free water at 0.5 μg/μL. Nanoparticles are
formulated by mixing the amino lipids with plasmid DNA for 30 min
in nuclease-free water at prespecified N/P ratios. The size and zeta
potential of the nanoparticles were analyzed using an Anton Paar Litesizer
500 instrument (Anton Paar USA Inc., Ashhland, VA) in nuclease free
water.
The encapsulation of CRISPR/cas9 plasmids in the nanoparticles
was assessed by gel electrophoresis. Lipid/plasmid DNA nanoparticles
(4 μL) and 4 μL of loading dye (Promega, Madison, WI)
and 16 μL nuclease free water were mixed. The mixture (20 μL)
was loaded onto a 0.7% agarose gel containing ethidium bromide. The
gel was submerged in 0.5× Tris/Borate/EDTA (TBE) buffer and run
at 100 V for 25 min. Plasmid DNA bands were visualized using GelDoc
XRS (Bio-Rad, Hercules, CA).
plasmids were purchased from the Addgene repository. (Addgene plasmids
#78535 and 78547).38 (link) Two sgRNAs were selected
to target eGFP sequence (sgRNA1: GAGCTGGACGGCGACGTAAACGG;
sgRNA2: CAGAACACCCCCATCGGCGACGG). The
amino lipid carriers were dissolved in ethanol at a stock concentration
of 2.5 mM, while the plasmids of CRISPR/Cas9 system were reconstituted
in nuclease-free water at 0.5 μg/μL. Nanoparticles are
formulated by mixing the amino lipids with plasmid DNA for 30 min
in nuclease-free water at prespecified N/P ratios. The size and zeta
potential of the nanoparticles were analyzed using an Anton Paar Litesizer
500 instrument (Anton Paar USA Inc., Ashhland, VA) in nuclease free
water.
The encapsulation of CRISPR/cas9 plasmids in the nanoparticles
was assessed by gel electrophoresis. Lipid/plasmid DNA nanoparticles
(4 μL) and 4 μL of loading dye (Promega, Madison, WI)
and 16 μL nuclease free water were mixed. The mixture (20 μL)
was loaded onto a 0.7% agarose gel containing ethidium bromide. The
gel was submerged in 0.5× Tris/Borate/EDTA (TBE) buffer and run
at 100 V for 25 min. Plasmid DNA bands were visualized using GelDoc
XRS (Bio-Rad, Hercules, CA).
