TY - JOUR
T1 - Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs
AU - Li, Pan P.
AU - Margolis, Russell L.
N1 - Funding Information:
We thank Dr. Xiao-bing Zhang for the kind gift of the pEF-BCL-XL plasmid, Dr. Feng Zhang for the kind gift of the pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) plasmid, Dr. Mahmoud Pouladi for the kind gift of the pJOP-HTT-HR18Q plasmid, and Dr. Tobias Cantz for the kind gift of the AAT-PB-CG2APtk plasmid. This work was supported by the National Institutes of Health grants NS112796, NS112687, and NS100783, the National Ataxia Foundation, and the ABCD Charitable Trust.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.
AB - Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.
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U2 - 10.1038/s41598-021-89312-2
DO - 10.1038/s41598-021-89312-2
M3 - Article
C2 - 33972655
AN - SCOPUS:85105549543
SN - 2045-2322
VL - 11
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 9865
ER -