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, 20 (12), 974-982

Fast and Cloning-Free CRISPR/Cas9-mediated Genomic Editing in Mammalian Cells

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Fast and Cloning-Free CRISPR/Cas9-mediated Genomic Editing in Mammalian Cells

Paul T Manna et al. Traffic.

Abstract

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

Keywords: CHoP-In; CRISPR; endogenous tagging; genome editing; mammalian cells.

Figures

Figure 1
Figure 1
Genome editing by CHoP‐In. CHoP‐In genome editing relies utilises NHEJ‐mediated integration of a PCR‐generated donor into a CRISPR/Cas9‐induced DSB. A, To achieve this, two constructs must be prepared after identifying the genomic gRNA and PAM site. To introduce a DSB at the desired locus, a vector such as pX330 encoding both the gRNA and the Cas9 nuclease is constructed. Additionally, a CHoP‐In integration donor is produced by PCR, consisting of the desired integration fragment flanked by the same gene specific gRNA and PAM sites in the PCR primers. Importantly, the gRNA and PAM sites flanking the integration donor must be in the reverse orientation with respect to genomic locus as this prevents reconstitution and re‐cleaving of the sites following integration. B, The whole process can be completed in approximately 1 week, giving a mixed population of edited cells with minimal hands‐on time when compared with HDR mediated approaches
Figure 2
Figure 2
Generation and characterisation of an EmGFP‐RAB5C HeLa line. A, Sequence encoding an N‐terminal EmGFP tag was integrated into the endogenous RAB5C locus by CHoP‐In. B, A sense strand gRNA and PAM site was selected immediately upstream of the RAB5C start codon and CHoP‐In primers were designed to amplify DNA encoding EmGFP, flanked by the necessary gRNA and PAM sites for intracellular cleavage and NHEJ‐mediated integration. C, Following transfection with px330‐RAB5C together with PCR donor fragments consisting of a frame corrected EmGFP tag without any Cas9 recognition site (no recognition seq.) or a full CHoP‐In donor fragment (ChoP‐In), cells were analysed and sorted by flow cytometry. WT cells are untransfected HeLa. Data are shown as FACS plots from individual experiments as well as mean data (+/− SD) from three independent experiments. D, Fluorescence microscopy revealed EmGFP signal (green) in cells from this mixed population which colocalised well with Alexafluor‐555 labelled endocytic tracer transferrin (red), following uptake of the marker for 15 minutes in order to label early endosomes (scale bar equals 10 μm). E, Immunoblotting with an antibody against RAB5C revealed the presence of a higher molecular weight band corresponding to the EmGFP‐RAB5C fusion in lysates from the mixed population. F, Sanger sequencing of integration junctions showed the fidelity of NHEJ mediated knock‐in of EmGFP into the RAB5C locus
Figure 3
Figure 3
C‐terminal tagging of ATP6V1G1. A, ATP6V1G1 was tagged at its C terminus with EmGFP. B, An antisense strand targeting gRNA and PAM site was selected which would create a DSB slightly upstream of the endogenous stop codon. EmGFP was amplified with CHoP‐In primers encoding sense orientation gRNA and PAM sites. C, Good colocalisation of EmGFP signal with the endolysosomal marker Magic Red was seen in a flow cytometry isolated EmGFP positive mixed population (scale bar equals 5 μm). D, Immunoblotting with an antibody against ATP6V1G1 confirmed expression of higher molecular weight, EmGFP‐tagged ATP6V1G1 from its endogenous locus. E, Off target expression of EmGFP was assessed by examining mixed populations of cells for aberrant GFP localisation
Figure 4
Figure 4
Internal tagging of AP2M1 and AP1G1. A, To create an internal AP2M1‐EmGFP fusion, a gRNA and PAM site was selected in exon 7 of the gene to generate an in‐frame insertion of EmGFP into the C‐terminal μ‐homology domain of the protein when expressed. B, Following isolation by flow cytometry, an EmGFP positive mixed population showed clear punctate plasma membrane EmGFP signal characteristic of endogenous AP2M1. C, To tag AP1G1 with mCherry, a gRNA and PAM site was selected in exon 20 of the gene in order to place mCherry within the flexible hinge region of the expressed protein. D, mCherry signal alone was at the limit of detection so the signal was amplified with an antibody against mCherry, revealing tubulovesicular perinuclear staining characteristic of endogenous AP1G1 Scale bars equal 10 μm

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