Plasmid Constructs for CRISPR Editing

The pmCherry_gRNA plasmid contained a U6-driven sgRNA scaffold and a cytomegalovirus (CMV)–driven pmCherry fluorescent protein. pmCherry_gRNA was a gift from E. Welker (Addgene plasmid #80457). pCMV_ABEmax_P2A_GFP (Addgene plasmid #112101) (24 (link)), NG-ABEmax (Addgene plasmid #124163) (28 (link)), pCMV-PE2-P2A-GFP (Addgene plasmid #132776) (22 (link)), and pU6-pegRNA-GG-acceptor (Addgene plasmid #132777) (22 (link)) were gifts from D. Liu. The N-terminal ABE and C-terminal ABE constructs were adapted from Cbh_v5 AAV-ABE N terminus (Addgene plasmid #137177) (29 (link)) and Cbh_v5 AAV-ABE C terminus (Addgene plasmid #137178) (29 (link)) and synthesized by Twist Bioscience and GenScript. The pSpCas9(BB)-2A-GFP (PX458) plasmid used for the generation of isogenic ∆Ex51 iPSCs was a gift from F. Zhang (Addgene plasmid #48138) (56 (link)). Cloning of sgRNAs was done using NEBuilder HiFi DNA Assembly (NEB) into restriction enzyme–digested destination vectors.

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Chemello F., Chai A.C., Li H., Rodriguez-Caycedo C., Sanchez-Ortiz E., Atmanli A., Mireault A.A., Liu N., Bassel-Duby R, & Olson E.N. (2021). Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing. Science Advances, 7(18), eabg4910.

Publication 2021

pCMV_ABEmax_P2A_GFP NG-ABEmax pU6-pegRNA-GG-acceptor Cbh_v5 AAV-ABE N terminus Cbh_v5 AAV-ABE C terminus pSpCas9(BB)-2A-GFP NEBuilder HiFi DNA Assembly

Cytomegalovirus Gifts Ipscs Plasmid Protein Restriction enzyme Vectors

Corresponding Organization : The University of Texas Southwestern Medical Center

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Variable analysis

independent variables

PmCherry_gRNA plasmid
PCMV_ABEmax_P2A_GFP
NG-ABEmax
PCMV-PE2-P2A-GFP
PU6-pegRNA-GG-acceptor
N-terminal ABE and C-terminal ABE constructs
PSpCas9(BB)-2A-GFP (PX458) plasmid

dependent variables

Cytomegalovirus (CMV)–driven pmCherry fluorescent protein
ABE activity
PE2 activity
Generation of isogenic ∆Ex51 iPSCs

control variables

U6-driven sgRNA scaffold

positive controls

None specified

negative controls

None specified

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