TY - JOUR
T1 - An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue
AU - Jin, Jiankang
AU - Xu, Yan
AU - Huo, Longfei
AU - Ma, Lang
AU - Scott, Ailing W.
AU - Pizzi, Melissa Pool
AU - Li, Yuan
AU - Wang, Ying
AU - Yao, Xiaodan
AU - Song, Shumei
AU - Ajani, Jaffer A.
N1 - Funding Information:
This study was supported in part by National Institutes of Health (NIH)/ National Cancer Institute (NCI) awards CA129906, CA127672, CA138671, and CA172741 and by Department of Defense grants CA150334 and CA162445 to J.A.A. and CA170906 and CA160433 to S.S. (J.A.A. = Dr. Jaffer A. Ajani; S.S. = Dr. Shumei Song). We declare that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study was supported by MD Anderson Cancer Center?s Flow Cytometry Facility. We thank Dr. Chang-Ru Tsai from the Department of Genetics at MD Anderson Cancer Center for providing a very informative critique of the manuscript. Thanks are extended to Dr. Laura L. Russell of MD Anderson Cancer Center?s Scientific Publications, Research Medical Library for language support.
Publisher Copyright:
© 2020 Jin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2020/2/1
Y1 - 2020/2/1
N2 - A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.
AB - A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.
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U2 - 10.1371/journal.pone.0228910
DO - 10.1371/journal.pone.0228910
M3 - Article
C2 - 32053639
AN - SCOPUS:85079335014
SN - 1932-6203
VL - 15
JO - PloS one
JF - PloS one
IS - 2
M1 - e0228910
ER -