Streamlined method for characterizing the variety of indels introduced by genome editing technologies: Ideal foridentifying indels introduced by CRISPR/Cas9 genome editing in a cell population
A complete kit containing all of the components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis: The Guide-it Indel Identification Kit includes reagents for PCR amplification directly from cells and for ligation-free cloning in15 minutes
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system is the newest tool for targeted genome editing. This genome editing technique is the most flexible method so far. This system is based on two key components that form a complex: the Cas9 endonuclease and a target-specific RNA (single guide RNA or sgRNA) that guides Cas9 to the genomic DNA target site. Double strand breaks that are introduced to genomic DNA by Cas9 nuclease can be repaired via the homologous recombination (HR) or nonhomologous end joining (NHEJ) cellular repair mechanisms. HR requires an identical (or nearly identical) DNA template to repair the break, and results in full correction of the DNA cleavage. However, DNA damage is more commonly repaired by NHEJ. In this case, cellular machinery ligates the two broken ends of the DNA without a template donor. This process is more error prone than HR, and ligation-free cloning system (Figure1,stepoften insertions and deletions (indels) are 2). After introduction into optimized Stellar incorporated, resulting in a DNA sequence that competent cells, colony PCR is used to amplify differs from the wild-type sequence.
Figure 1. Workflow for the guide-it Indel Identification Kit
A STREAMLINED METHOD FOR INDEL IDENTIFICATION
Any cell in a population has the ability to correct endonuclease cleavage by either HR or NHEJ, resulting in a variety of DNA sequences at the target site in different cells. To identify the range of indels at the genomic DNA target site in a cell population after editing, we developed a streamlined workflow to amplify, clone, and analyze target sites by DNA sequencing (Figure 1).
This protocol uses a small sample of the cell population as a template for direct PCR amplification of the genomic region containing the endonuclease target site. The use of Terra PCR Direct Polymerase allows amplification directly from crude cell lysates without the need for genomic DNA purification (Figure 1, step 1). The resulting PCR pool of DNA fragments, containing different mutations at the target site, are cloned into a pUC19 vector using In-Fusion Cloning, a ligation-free cloning system (Figure 1, step 2). After introduction into optimized Stellar competent cells, colony PCR is used to amplify the inserts (Figure 1, step 3). Finally, DNA sequencing of the amplified inserts is used to identify indels (Figure 1, step 4).
Figure 2. Flow cytometry analysis of HT1080-AcGFP1 cells. Cells were analyzed before (red line) and after (green) transfection with plasmids encoding Cas9 and an AcGFP1-targeting sgRNA.
KNOCKOUT OF ACGFP1 USING CRISPR/CAS9 GENOME EDITING
CRISPR/Cas9 genome editing technology was applied to disrupt a single copy of AcGFP1 integrated in the genome of HT1080 cells (HT1080-AcGFP1). These cells exhibit strong green fluorescence (Figure 2, red trace). Therefore, AcGFP1 knockout can be assessed by monitoring GFP expression by flow cytometry. In cells that were co-transfected with two plasmids, one expressing Cas9 and the other expressing an sgRNA targeting AcGFP1, a significant percentage of the population (67%) lost GFP expression, due to mutation of the AcGFP1-encoding gene (Figure 2).
The different indels created at the AcGFP1 locus in the cell population were identified using the Guide-it Indel Identification Kit (according to the protocol in Figure 1). Cells (2×105) from the transfected cell population
Figure 3. PCR product amplified from the Cas9 sgRNA target sequence. Lane M: Marker; Lane 1: Amplified AcGFP1 target site (* indicates location of PCR product).
Figure 4. Agarose gel of colony PCR products from eight clones. Lane M: Marker; Lane 1–8: Individual clones.
Figure 5. Sequencing results of the AcGFP1 target sequences obtained from eight different clones.
were collected and used directly as a template for PCR amplification of the genomic AcGFP1-encoding gene targeted by Cas9/sgRNA. The amplified genomic DNA fragments (Figure 3, lane 1) contain a mixture of the different indels that occurred via NHEJ repair at the AcGFP1 locus following double strand breakage. The PCR products were cloned into a pre-linearized pUC19 vector using In-Fusion Cloning. Eight of the resulting bacterial clones were selected for colony PCR using Terra PCR Direct Polymerase with primers that amplified the insert.
The amplified DNA was analyzed on an agarose gel, and all colonies showed a band of the correct size (Figure 4). The PCR products were sequenced using the provided sequencing primers. The obtained DNA sequence was aligned with the wild-type AcGFP1 sequence (Figure 5). The sequences showed a high rate of an inserted “T” at the Cas9/sgRNA cleavage site in the different clones. One clone had a four nucleotide deletion immediately upstream of the PAM site. Only one clone contained the wild-type AcGFP1 sequence (Figure 5).
Figure 6. Flow cytometry analysis of HeLa cells, labeled with and without an anti-CD81-FITC antibody
Figure 7. Flow cytometry analysis of HeLa cells labeled with the anti-CD81-FITC antibody after transfection with Cas9 and sgRNA targeting the CD81 gene.
Disruption of CD81 using CRISPR/Cas9 Genome Editing
CD81 is a membrane receptor expressed in most cell types. Using a FITC-tagged antibody against CD81, expression can be monitored by flow cytometry (Figure 6). The endogenous CD81 gene was targeted for CRISPR/Cas9 genome editing by transfecting HeLa cells with two plasmids encoding Cas9 and a CD81-targeting sgRNA, respectively. Five days after transfection the cells were harvested, labeled with an anti-CD81-FITC antibody, and analyzed by flow cytometry (Figure 7). The analysis indicated that CD81 expression was lost in approximately 51% of the cell population.
Figure 8. PCR products from amplification of the targeted CD81 site from the cell population analyzed in Figure 7. Lane M: Marker;
Lane 1: Amplified CD81 target site (* indicates location of PCR product).
The Guide-it Indel Identification Kit was used to characterize the nature of the indels created by Cas9/sgRNA in the cell population. Cells from the same population analyzed in Figure 7, which contained CD81 expressing and non-expressing cells, were used as template for direct PCR amplification of the targeted CD81 genomic site (Figure 8).
Figure 9. Agarose gel of colony PCR products directly from six clones. Lane M: Marker; Lane 1–6: Individual clones.
Figure 10. Sequence analysis of six clones obtained from cells transfected with Cas9/sgRNA targeting the CD81 gene
After cloning the PCR products, six bacterial clones were picked for colony PCR amplification of the CD81 target sequence using Terra PCR Direct Polymerase. The amplified DNA was analyzed on an agarose gel, and all colonies showed a band of the correct size (Figure 9). The amplified PCR products were sequenced using the provided sequencing primer, and the sequencing data were aligned to the wild-type CD81 sequence (Figure 10). The obtained sequencing data from the different clones showed a wide variety of deletions and insertions at the targeted CD81 gene, emphasizing the variation in the outcome of NHEJ repair between individual cells.