Gene Editing by CRISPR/Cas9 and The Importance of Sanger Sequencing To Power Edit Confirmation

The last decade has witnessed the rapid development and advancement of gene editing technologies. Methods such as CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) have opened a world of possibilities for researchers to not only understand the genetic basis of several human pathologies, but also develop therapeutic strategies against both inborn as well as infectious diseases such as HIV, SARS-CoV-2 and others. Given the implications of DNA modification on cell function, it is important to validate and understand the nature of such genetic edits using accurate and reliable tools.

The Advent of Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)

Although other genome editing methods, such as TALENs (Transcription activator-like effector nucleases) and ZFNs (zinc finger nucleases), have been in use, CRISPR is newer and simpler gene editing technology. In 2020, Emmanuelle Charpentier and Jennifer Doudna, won the Nobel Prize in Chemistry for biochemically characterizing the Cas9-mediated DNA modification system.

In its native role, CRISPR is an adaptive defense mechanism used by bacteria and other prokaryotes to protect against viruses and plasmid challenges. Briefly, it involves integration of short nucleic acid sequences of the foreign DNA (Spacer) into the host genome adjacent to an array of repetitive elements (Repeats). The Spacer sequences, when transcribed and processed into small RNAs, form a functional complex with a nuclease (Cas9) which recognizes and digests the incoming foreign DNA. This mechanism was subsequently adapted for eukaryotic genomes and was first shown to modify human and mice cells in 2013 [1].

To target a gene, a guide RNA (gRNA) is designed that bears complementarity to the region of interest. This gRNA then directs the CRISPR/Cas9 nuclease complex to the specific sequence. The cut made by the nuclease is then the site that gets modified, either by non-homologous end joining or by template directed repair. Unlike TALENs, which require a unique transcription activator-like (TAL) effector protein for each target DNA sequence, CRISPR only requires switching guide RNA sequences, making the latter much easier for gene editing applications.

Applications of CRISPR in Disease Research

Genetic and Infectious Diseases

Muscular Dystrophy

Several applications of CRISPR have been demonstrated for the research of debilitating and life-threatening diseases. For instance, Bengtsson and colleagues showed the application of the system in correcting a Duchenne muscular dystrophy (dmd) gene mutation in an animal model of the disease through muscle specific expression of CRISPR/Cas9 [2]. Gene-edited mice exhibited a robust expression of dystrophin in the muscles and amelioration of the disease-associated phenotype.

A diagnostic test with high sensitivity and specificity is the key to solving this problem. Sensitivity and specificity are measures of a diagnostic test’s ability to correctly classify an animal as having a disease or not having a disease.

HIV

In addition to genetic diseases, CRISPR has also shown promise as a potential treatment strategy for HIV infection. A major bottleneck for achieving HIV cure is that the virus integrates into host genome and therefore infected patients must be on lifelong antiviral treatment to suppress virus replication. Recent studies in macaques showed that Cas9 was effective in eliminating the proviral DNA of simian immunodeficiency virus (SIV), a virus closely related to HIV, from infected blood cells as well as tissue reservoirs of the virus and preventing its replication [3].

CRISPR/Cas9 Continuous Improvement

The CRISPR/Cas9 system continues to be improved and adapted to new problems. Several new nucleases, including RNA-guided nucleases such as Cas12a, Cas12b etc. have been discovered and optimized to expand the selection of targets as well as features such as small size and protospacer adjacent motif (PAM) sequences.

CRISPR for Genome Modification in Research of SARS-CoV-2 and Influenza A

One of the powerful applications of these nucleases is to direct targeting and modification of RNA virus genomes, including the genomes of several respiratory pathogens. Abbott and colleagues recently published a proof-of-concept study using this strategy to digest RNA genome from SARS-CoV-2 and Influenza A virus to inhibit viral replication in human lung epithelial cells [4].

Importance of Evaluating Gene Editing Efficacy in Genome Modification

When performing a genome modification, several factors contribute to the success of the edit.

Different guide RNAs have different targeting efficacies.
Different methods are available for delivering CRISPR/Cas9 complexes into cells (for complete list, see product and services in our catalog).
Different cell types have different repairing efficiencies.
Finally, the repair mechanism itself produces a heterogenous mixture of sequences around the target site.

An important element of genome editing is therefore to determine the efficiency of an edit under the experimental conditions.

Sanger Sequencing in Genome Modification Experiments

Sanger sequencing is a useful tool for determining editing efficiencies in a genome modification experiment. Here, bulk DNA from the post-treatment primary cell population can be extracted and subjected to Sanger sequencing. This primary population will be a mixture of cells that include both non-edited and a heterogeneous mix of edited sequences.

By examining the Sanger sequencing traces from the mixed population, the abundance of edited and non-edited genomes can be seen. Although exact sequences in the population might not be discernable, the relative amount of all non-wild-type sequences helps determine the efficiency of the genome modification.

If the editing efficiency is high enough, the next step in a genome editing experiment is to isolate the cells that have the desired edit. Typically, this is done by limiting dilution of the primary culture to single cells which are then clonally expanded. Each clonal culture is then checked for genetic modifications around the editing site by Sanger sequencing. This will give the exact sequence of the population of cells, and can then be used for downstream applications.

Simplifying Sanger Sequencing Analysis Using New Tools

Although other methods can be used to determine editing efficiency, there are several advantages of Sanger sequencing. Mismatch cleavage assays rely on enzymes to cleave heteroduplex DNA at mismatches introduced on one of the strands during the cellular repair process. While these methods can detect the INDEL variants, they lack sensitivity and do not provide any information related to the exact sequence of the mutation. In addition, they are unable to detect homozygous mutants.

The T7E1 endonuclease in particular does not recognize single nucleotide polymorphisms well [5]. Indirect methods such as evaluation of gene or protein expression demonstrate functional impact of the gene edit(s) but do not provide any indications regarding the type of mutation. And NGS-based methods can be expensive, time-consuming and difficult to analyze. Sanger sequencing, on the other hand, remains the gold standard for validation as it enables precise identification of the gene modification in an easy, reliable and sensitive manner.

Applied Biosystems SeqScreener Gene Edit Confirmation Software

More Information and Better Resolution

Although the results of a Sanger sequencing trace can be visually inspected for general idea of efficiency, more information and better resolution can be obtained using software tools. To expedite analysis of such data, a new gene editing software solution (drum roll please), “Applied Biosystems SeqScreener Gene Edit Confirmation App” by ThermoFisher Scientific, was recently developed.

Feature-Rich Software

Range and Frequency

SeqScreener enables determination of both the range as well as frequency of the INDEL mutations in cultures of the primary or secondary engineered cells. The software can analyze the gene editing efficiency as well as identify and rank candidate guide RNAs.

Visualization

The software has a Plate View option that enables visualization of insertion or deletion mutations in one glance. It also provides the editing percentage and edit diversity in the cell population. This is especially helpful for high throughput experiments that may involve multiple gRNAs, cell types or transfection methodology evaluation.

Efficiency

SeqScreener has features not only for determining the efficiencies of INDELs created by non-homologous end-joining, it also can determine the efficiency of template-directed edits, such as changing a SNP.

High Throughput

Finally, SeqScreener was designed to be able to analyze up to 96 samples at once – this batch uploading feature facilitates analysis in high-throughput labs.

CRISPR/Cas9 and Sanger Sequencing for Gene Editing Confirmation

Gene editing technologies such as CRISPR hold great promise in medicine and are only beginning to be explored as viable treatment options for several diseases. With the heightened interest in the field, awareness around tools that enable researchers to confirm genomic modifications with confidence is important. Sanger sequencing with its several advantages is one of the most reliable methods to validate nucleotide changes with high resolution. With an added benefit of the SeqScreener software, data interpretation from Sanger sequencing is no longer an arduous task.

References

Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.
Bengtsson, N.E., et al., Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun, 2017. 8: p. 14454.
Mancuso, P., et al., CRISPR based editing of SIV proviral DNA in ART treated non-human primates. Nat Commun, 2020. 11(1): p. 6065.
Abbott, T.R., et al., Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell, 2020. 181(4): p. 865-876 e12.
Vouillot, L., A. Thelie, and N. Pollet, Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda), 2015. 5(3): p. 407-15.