November 1, 2017 (Vol. 37, No. 19)
Streamlining the CRISPR Workflow with a Heteroduplex Cleavage Assay
The development of CRISPR/Cas9 revolutionized researchers’ ability to edit the genome at will. However, the nature of CRISPR edits is not entirely controllable or predictable. This uncertainty is due to the mechanism by which the edits are integrated into the genome.
When CRISPR induces a double-stranded break (DSB) in DNA, the break is repaired via random one- or two-base pair (bp) indels through nonhomologous end joining (NHEJ). In diploid cells, these mutations can take on one of three zygosities: monoalleic, where only one allele is mutated; diallelic heterozygous, where both alleles are mutated differently; or diallelic homozygous, where both alleles are mutated identically.
These different outcomes require researchers to screen their cell lines for the desired zygosity of the mutation. Next-generation sequencing (NGS) is the gold standard for zygosity screening, but the process is costly and time-consuming, particularly if there are many cell lines that require screening.
This tutorial will introduce a new screening method that uses Accucleave™ T7CE, a T7 endonuclease I (T7EI)-based heteroduplex cleavage assay that specifically screens out cell lines with undesired zygosity prior to NGS. The tutorial will also introduce new statistical models that aid in determining the zygosity of cell lines, thus providing a rapid screening step that reduces the number of cell lines that must undergo sequencing, saving time and money.
How the Assay Works
AccuCleave T7CE screens zygosity by identifying and cleaving DNA mismatches between corresponding alleles. If the mutation is monoallelic, diallelic homozygous, or diallelic heterozygous, T7EI will cleave the resulting heteroduplex, yielding a predictable fragmentation pattern. The relative concentration of cleaved fragments can be used to estimate the zygosity of monoclonal diploid cell lines.
The assay will not cleave the homoduplexes formed when diallelic homozygous mutations occur, rendering them indistinguishable from wild-type (WT) cells. Therefore, following PCR amplification, but prior to heteroduplex formation, it is necessary to introduce WT DNA to the mutated DNA at a 50/50 ratio to accurately measure the occurrence of all three possible zygosities in each cell line (Figure 1).
Figure 1. A representation of the possible zygosities of a nonhomologous end joining (NHEJ)-induced mutation following a CRISPR edit in a diploid cell, and the resulting duplexes when this DNA is mixed in a 50/50 ratio with wild-type DNA. Also shown are the theoretical cleavage percentages following the application of AccuCleave T7CE to each sample.
The AccuCleave T7CE Kit is used after a PCR-amplified DNA sample is denatured and reannealed to allow heteroduplex formation. First, samples are mixed with the T7EI master mix and incubated at 37°C for 30–60 minutes, which allows the endonuclease time to cleave heteroduplexes. Then, the mixture is diluted with Tris-EDTA and analyzed using the Fragment Analyzer CRISPR Discovery Gel Kit from Advanced Analytical Technologies. Finally, the percentage of cleaved duplexes is calculated using the CRISPR plugin for PROSize® Data Analysis Software.
The percent cleaved is calculated by measuring the relative amount of cleaved versus noncleaved fragments following T7EI digestion. Equation 1 describes how the percent cleaved is calculated: CF1 = the concentration of one of the resultant fragments following cleavage; CF2 = the concentration of the other resultant fragment; and CA = the concentration of the initial, noncleaved amplicon. When one is working with pooled cell lines, the percent mutated can be calculated from the percent cleaved using Equation 2, allowing for mutation detection as low as 5% mutated.
For monoclonal diploid cell lines, the zygosity can be predicted from the percent cleaved. When WT DNA is mixed in a 50/50 ratio with monoallelic DNA, the amount of mutated alleles in the whole sample will be 25%. When DNA from a cell line with a diallelic heterozygous mutation is mixed with WT DNA, 25% of the strands will harbor mutation A, 25% will have mutation B, and 50% will be WT.
Finally, when WT DNA is mixed with an equal portion of diallelic homozygous DNA, 50% of the total DNA will have the mutation. Based on composition of each of these pools, the theoretical probabilities that heteroduplexes will form are, with respect to zygosity, 37.5% for cell lines with monoallelic mutations, 61.5% for cell lines with diallelic heterozygous mutations, and 50.0% for cell lines with diallelic homozygous mutations (Figure 1). These theoretical percentages serve as landmarks that allow researchers to derive the zygosity of a mutation in a particular cell line based on the T7EI cleavage frequency.
Equation 1 and 2
Assessing the Assay’s Validity
In a proof-of-method study, the AccuCleave T7CE Kit was used to assess the zygosity of several model DNA strands, each containing a multitude of single-nucleotide polymorphisms and indels of varying lengths.1 After PCR amplification, the DNA sequences were digested using the kit. The researchers tested the kit on amplicons of varying lengths, and found that only the 600-bp amplicon exhibited cleavage rates within 5% of the theoretical value of each zygosity (Figure 2).
In the 600-bp amplicon, the endonuclease was able to cleave all single-base mismatches along with 1-, 2-, and 10-bp indels within 5% of the expected theoretical cleavage frequency for each mutation (Figure 3). As all zygosities fell within 5% of their theoretical percent cleaved landmarks, there was no overlap for monoallelic, diallelic homozygous, and diallelic heterozygous mutations, making them easily distinguishable (Figure 2). This method was further confirmed in CRISPR-edited rice.
Figure 2. A comparison of cleavage percentage for different amplicon lengths. The 600-bp amplicon was cleaved at rates that were consistent with the theoretical cleavage percentages (that is, 37.5% for monallelic mutations, 50% for diallelic homozygous mutations, and 62.5% for diallelic heterozygous mutations).
Figure 3. A comparison of cleavage percentage for different mutation types. All mutations were cleaved at values consistent with the theoretical cleavage percentages (that is, 37.5% for monallelic mutations and 50% for diallelic homozygous mutations).
Conclusion
As CRISPR is used more in diploid cells, such as in induced human pluripotent stem cells and cells from rodent models, it will become increasingly critical to use an assay that can prescreen cell lines for zygosity and reduce the need to perform NGS. AccuCleave T7CE, a T7EI-based heteroduplex cleavage assay, accurately and reliably cleaves heteroduplexes, and an analysis of the resulting cleaved fragments can be used to characterize the zygosity of mutations in the sample. This tool can help researchers identify cell lines with their desired mutation prior to NGS, saving them time and money, and streamlining their CRISPR workflows.
Steve Siembieda ([email protected]) is vice president of commercialization, and Kyle Luttgeharm, Ph.D., works as an application scientist at Advanced Analytical Technologies.
Reference
1. K.D. Luttgeharm, K.S. Wong, and S. Siembieda, “Heteroduplex Cleavage Assay for Screening of Probable Zygosities Resulting from CRISPR Mutations in Diploid Single Cell Lines,” BioTechniques 62(6), 268–274 (June 2017).
