An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue

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.

A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries

“…The cloned library was then packaged into lentivirus and used to transfect HeLa GFP-LC3 cells. Sorting of the transfected and untransfected cells by FACS showed that ∼15% of the transfected cells lost GFP expression (Figure 4B , Supplemental Figure S3), comparable to previous studies looking at CRISPR efficiency ( 28 ). Sequencing of the incorporated spacers and GFP mutations in each pool showed that all eight potential spacers could be found in the GFP-negative pool and mutations occurred at all four sites (Figure 4C ).…”

SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries

“…The cloned library was then packaged into lentivirus and used to transfect HeLa GFP-LC3 cells. Sorting of the transfected and untransfected cells by FACS showed that approximately 15% of the transfected cells lost GFP expression ( Figure 3B, Supplemental Figure 2), comparable to previous studies looking at CRISPR efficiency 22 .…”

FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells

“…To overcome both hurdles, we used modified cDNAs for gene rescue. In case of CR1SPR/Cas9 gene knockout, because Cas9 cleaves where gRNA binds, one or a few nucleotides in the gRNAS binding site of the CD44v6 and YB-1 were modified by three nucleotides, while the amino acid sequences remained unchanged as described previously in generating shRNA resistant constructs and gRNA rescue constructs [ 57 , 58 , 173 ]. Total cell lysates were examined by Western blot analysis for the indicated proteins and β-tubulin or β-actin were used as internal standards.…”