Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation

Abstract

Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.

Keywords: Biotechnology and bioengineering; CRISPR; Cell-based Assays.

Graphical abstract

Figure 1

Target1 region PCR primers, guide sequence (gRNA1), protospacer adjacent motif (PAM), and cut site are indicated.

Figure 2

In vitro cleavage efficiency of RNPs (A) Representative electropherogram and virtual gel showing the visualization of DNA substrate corresponding to uncleaved PCR product of Target 1 on an Agilent 5200 Fragment Analyzer using a 1–6000 bp fragment kit. Size and molarity are indicated (teal rectangles). RFU: Relative Fluorescence Units, LM: Lower Marker, UM: Upper Marker. (B) Representative electropherogram and virtual gel showing the visualization of an IVC reaction of Target1 on an Agilent 5200 Fragment Analyzer using a 1–6000 bp fragment kit. Size and molarity of DNA fragments resulting after cleavage of DNA substrate are indicated: cleaved fragments (red and blue rectangles) and uncleaved fragment (black rectangles). RFU: Relative Fluorescence Units, LM: Lower Marker, UM: Upper Marker. (C) Cleavage efficiency is calculated as the fraction of DNA substrate that is cleaved (i.e., the sum of the molar amounts of cleaved fragments over the sum of the molar amounts of cleaved and uncleaved fragments). Cleavage efficiency calculation for IVC reaction in (A) is shown. (D) Cleavage efficiency of gRNA1 and gRNA2 RNPs formed at Cas9 to gRNA molar ratios of 1 to 9 and 1 to 2. Two independent IVC reactions performed in duplicate are shown for each ratio. Data are represented as mean ± SEM.

Figure 3

Transfection efficiency of ST cells (A) ST cells were nucleofected with pmaxGFP using four pulse codes and the SF Nucleofector Solution. Representative percentages of GFP+ cells for each pulse code measured 24 h post-nucleofection using a Nexcelom Cellometer Spectrum Image Cytometry System are shown. (B) Representative microscopy images of ST cells transfected as in (A). For each pulse code, one field of view with phase, fluorescence and overlay images are shown. Scale bar, 200 mm.

Figure 4

Analysis of CRISPR-Cas9 genome editing using ICE (A) ICE tool workflow. (B and C) Representative ICE tool outputs for Target1 (B) and Target2 (C). Top: alignment of control and edited sequence traces generated using Target1 Reverse primer (B) and Target2 Reverse primer (C) to the provided guide sequence (underlined). Dotted line represents inferred cut site.

Figure 5

Editing efficiency of RNPs Editing efficiency expressed as indel percentage of gRNA1 and gRNA2 RNPs formed at a Cas9 to gRNA molar ratio of 1 to 2. Two independent nucleofection reactions are shown. Data are represented as mean ± SEM.