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Tech Note: Raising the Bar on Single-Cell and Ultra-Low Input mRNA Sequencing

TECH NOTE

Successful Identification of Monoallelic and Biallelic Mutants after CRISPR/Cas9 Gene Editing

Overview

The CRISPR/Cas9 system is a powerful tool for targeted gene editing, in which a single guide RNA (sgRNA) directs a Cas9 nuclease to a specific genomic locus. Cas9 cleaves the gene of interest and creates a double-stranded break (DSB) in the DNA, which can be repaired by non-homologous end joining (NHEJ). As NHEJ is an error-prone DNA repair process, insertions and deletions (indels) are often introduced into the gene, resulting in frameshifts and potential loss of gene function.

It is often necessary to determine if there are indels in one or both alleles (monoallelic or biallelic mutants, respectively). To achieve functional knockout, both copies of the gene need to be mutated. This is especially important for certain applications such as disease modeling or the development of transgenic animals (1, 2). In some cases, a monoallelic mutation can be useful or necessary, such as when modeling specific diseases. Therefore, it is necessary to confirm the exact genotype of cells following CRISPR/Cas9 gene editing. Traditionally, this has involved lengthy subcloning and sequencing steps. However, a streamlined protocol with the ability for direct assessment can save time while providing high accuracy.

An in vitro cleavage assay for genotype determination

The Guide-it Genotype Confirmation Kit uses a Cas9/sgRNA-mediated in vitro cleavage assay for genotype determination. PCR amplicons containing the potentially edited genomic locus are used in a cleavage reaction set up with the same sgRNA as was used for the initial gene editing. The cleavage products are then separated on an agarose gel. If both alleles of the target site are wild type (wt), the Cas9/sgRNA complex will cleave the PCR amplicons, which would appear on the gel as two small DNA fragments. Conversely, if both alleles have indels (biallelic mutation), the Cas9/sgRNA complex will not cleave, and the gel will show only one large DNA fragment. If just one allele has an indel, as in the case of a monoallelic mutation, only the wt allele will be cleaved, resulting in one large fragment and two small fragments on the gel. In this manner, a particular cell's genotype can be directly assessed, without the need for molecular subcloning and subsequent sequencing.

Guide-it indel identification kit overview

Schematic of genotype determination by in vitro Cas9/sgRNA cleavage. The same sgRNA that was used for gene editing must be used in the Cas9/sgRNA in vitro cleavage reaction. Cleavage products are resolved on an agarose gel.

Accurate genotype determination of HEK 293 cells

The C4BPB gene encodes one of the two polypeptides for human plasma glycoprotein C4b-binding protein (C4BPB), and has a role in the complement pathway, which is key to innate immunity. In the following experiment, HEK 293 cells were treated with Cas9 and an sgRNA targeting C4BPB. Fifteen single-cell clones were isolated and the Guide-it Genotype Confirmation Kit was used for genotype determination, as shown in the figure below.

Experimental workflow of the Guide-it Genotype Confirmation Kit. Single-cell clones are generated from cells treated with Cas9 and a gene-specific sgRNA. Crude DNA extracts are prepared from the isolated clones, and the target gene is amplified by PCR. The resulting product is then used in an in vitro cleavage reaction with Cas9 and the same sgRNA that was used for the original gene editing experiment. The DNA fragments are then separated via agarose gel electrophoresis.

Following CRISPR/Cas9 editing, the C4BPB locus was PCR-amplified from crude DNA extracts. In vitro cleavage of the PCR products was set up with recombinant Cas9 nuclease and the same sgRNA that was used for gene editing. Control reactions (included in the kit) were included in the analysis. Results of agarose gel electrophoresis on the cleaved DNA fragments indicated that clones 1, 4, 10, and 12 were wt. Clone 2 was monoallelic (M), and clones 3, 5–9, 11, and 13–15 were biallelic (B).

Genotype determination by the Guide-it Genotype Confirmation Kit. Reactions for clones 1, 4, 10, and 12 resulted in two small fragments, and were therefore wt. The reaction for clone 2 resulted one large fragment and two small fragments, indicating it was monoallelic (M). Reactions for clones 3, 5–9, 11, and 13–15 all resulted in single large fragments, indicating they were biallelic (B).

In order to confirm the in vitro genotype assay, the C4BPB locus was subcloned for Sanger sequencing. For each of the four clones selected, the sequencing results corroborated the results of the Guide-it Genotype Confirmation Kit. As predicted, clone 2 was monoallelic, while clone 7 was heterozygous biallelic. Clones 9 and 11 were homozygous biallelic, representing biallelic deletion and insertion mutants, respectively.

Sanger sequencing corroborates results of the Guide-it Genotype Confirmation Kit. For each result, lowercase nucleotides represent the wt reference sequence. Clone 2 is monoallelic, although three different alleles were detected. It is possible that this result stems from copy number variation in the HEK 293 cell line. Sequencing indicated that clone 7 is a heterozygous biallelic mutant, and clone 9 and 11 are homozygous biallelic mutants.

Conclusions

The Guide-it Genotype Confirmation Kit is a streamlined, in vitro alternative to tedious subcloning and sequencing following CRISPR/Cas9 editing of target genes. This assay—performed directly from crude DNA extracts—allows monoallelic and biallelic mutations to be quickly and easily identified. Genotyping is simple yet accurate, further improving your gene modification workflow.

Methods

Disruption of the C4BPB gene in HEK 293 cells:

1 x 105 HEK 293 cells were seeded in 12-well plates one day prior to transfection. Cells were cotransfected with 2.5 µg of pGuide-it-ZsGreen1 plasmid encoding Cas9 and an sgRNA designed to target C4BPB using the Xfect Transfection Reagent.

Genotype determination assay

48 hours after transfection, single-cell clones were established by the dilution method. Crude DNA extracts were prepared by using the extraction buffer included in the Guide-it Genotype Confirmation Kit. Following extraction, the C4BPB locus was PCR-amplified using the Terra PCR Direct Polymerase Mix included in the kit. For downstream applications, 96-well replica single-cell plates were generated.

In vitro cleavage reactions were set up using the recommended protocol in the Guide-it Genotype Confirmation Kit, with PCR amplicons, 250 ng of the Guide-it Recombinant Cas9 Nuclease, and 50 ng of the sgRNA. The same sgRNA used during gene editing was used in this in vitro cleavage assay. Cleavage reactions were run on a 2% agarose gel.

Sanger sequencing of the C4BPB locus

The C4BPB locus was subcloned into pUC19 from single-cell HEK 293 clones using the Guide-it Indel Identification Kit. PCR fragments were sequenced using standard pUC19 primers included in the kit.

Reference:

1. Hai, et al. (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research 24:372–375.

2. Jao, et al. (2013) Efficient multiplex billalelic zebrafish genome editing using a CRISPR nuclease system. PNAS 110(34):13904–13909.

3. Rodriguez de Cordoba, et al. (1994). The gene coding for the beta-chain of C4b-binding protein (C4BPB) has become a pseudogene in the mouse. Genomics 21(3):501–509.

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