Abstract
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.
Highlights
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful gene editing tool capable of performing DNA cleavage with the help of guide RNAs and the constitutive expression of Cas9 [1] [2]
When V2mO was made into lentiviruses, with proper guide RNAs (gRNAs), i.e. RhoA-gRNA5, Gli1-gRNA4, and Gal3-gRNA1, they sufficiently knocked out RhoA, Gli1, and Gal3 genes in gastric cancer (GC) cell lines AGS and GT5 as visualized by short PCR electropherograms around gRNA binding regions and detected by Western blots
Gene editing efficiencies or knockout ratios of target cell pools could be estimated by direct Sanger electropherograms from Cas9-gRNAs transduced cell populations without the sequences of control or parental populations
Summary
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful gene editing tool capable of performing DNA cleavage with the help of guide RNAs (gRNAs) and the constitutive expression of Cas9 [1] [2]. Substantial numbers of gene knockout (KO) experiments using pLentiCrispr-V1 or pLentiCrispr -V2, or pLenti-Cas plus pLenti-GuidePuro (Version 3)[3, 4] have failed. Factors contributing to the failure include low titers of the lentiviral Cas, less-than-perfect gRNAs, inefficient selective markers for constitutive expression of Cas, difficult-to-transfect target cells (e.g., primary cells), and wildtype (WT) or WTlike clones overgrowing and taking over edited cell pools. Improved CRISPR/Cas9-mOrange-puro gene knockout and rescue design, data collection and analysis, decision to publish, or preparation of the manuscript
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