BioTechniques Explores Precise Genome Manipulation Sections and CRISPR-Based Diagnostics

Diagnostic technologies that leverage clustered regularly interspaced short palindromic repeats (CRISPR) offer considerable gene editing prowess. However, the CRISPR/Cas9 technology is better suited to gene inactivation than repair. Although targeting the Cas9 enzyme to a genomic sequence is relatively precise, the cell’s repair of the resulting double-stranded cut isn’t precise. Furthermore, as CRISPR/Cas9 repairs are mediated by a method known as non-homologous end-joining, these repairs are often muddied by small deletions or insertions.

Here, the life sciences journal BioTechniques examines CRISPR base editing and prime editing techniques, nucleic acid-based diagnostics, CRISPR-based methods for disease diagnosis, and the Cas13 enzyme.

Base Editing and Prime Editing

Most genetic diseases need gene correction instead of disruption. David Liu, a chemical biologist at Harvard University, has worked with his team to develop two approaches: base editing and prime editing. These approaches exploit CRISPR’s precise targeting while limiting Cas9’s ability to cut DNA at a specific site. As a result, both cut a single DNA strand, a safer, less disruptive process for cells.

Base editing combines a catalytically impaired form of Cas9 with an enzyme that encourages the chemical conversion of one nucleotide to another, such as adenine to guanine or cytosine to thymine. However, this method only makes specific base-to-base changes accessible. Base editing was first described in 2016 and is already progressing toward clinical use. Liu has founded Beam Therapeutics and received approval from the U.S. Food and Drug Administration to trial this approach on sickle-cell disease patients.

On the other hand, prime editing, a newer development, connects Cas9 to reverse transcriptase (a type of enzyme) and uses a modified guide RNA to include the desired edit in a genomic sequence. These components copy the guide RNA into DNA during a multistage biochemical process that replaces the targeted genome sequence. Prime editing hasn’t progressed as far as base editing, but improved iterations are emerging. For example, Hyongbum Henry Kim, a genome editing specialist at Younsei University College of Medicine, and his team have demonstrated that they can achieve up to 16% efficiency using this type of gene editing to correct retinal gene mutations in mice.

Meanwhile, Liu’s group has identified that prime machinery can aid the insertion of gene-sized DNA sequences into the genome. This can provide a better controlled, safer strategy for gene therapy. Although the process is relatively inefficient, even a minor repair can go a long way.

Nucleic-Acid-Based Diagnostics

Clinicians must diagnose diseases and select treatments quickly and accurately if they are to ensure optimum patient outcomes. Nucleic acid-based biomarkers associated with a disease are key to diagnostics because researchers can amplify DNA and RNA from trace amounts, enabling precise detection by pairing complementary nucleotides. Nucleic acid-based diagnostics have set a new standard for various acute and chronic conditions, especially those caused by infectious diseases. During outbreaks of infectious diseases, fast and precise nucleic-acid-based testing is essential to ensure adequate disease control, as proven by the COVID-19 pandemic.

Nucleic-acid-based diagnostics that rely on sequencing or quantitative polymerase chain reaction (qPCR) have been widely adopted, especially in clinical laboratories. The robustness, sensitivity, and versatility of PCR have made this technology one of the most popular for detecting RNA and DNA biomarkers. Although diagnostics based on the detection of nucleic acids are some of the most specific and sensitive, most assays require trained personnel and costly equipment.

Isothermal nucleic acid amplification avoids the need for thermal cyclers. But non-specific amplification can lead to lower detection specificity. Researchers can improve the specificity through additional readouts, especially oligo-strand-displacement probes, fluorescent probes, or molecular beacons. Researchers need technologies that team the low cost and ease of use of isothermal amplification with PCR’s diagnostic accuracy. In an ideal world, next-generation diagnostics should also have single-nucleotide specificity. This is essential to detecting mutations conferring resistance against antiviral drugs or antibiotics.

Fortunately, CRISPR-based diagnostics have the potential to meet these needs. There are many diverse CRISPR/Cas systems among the many species of archaea and bacteria. These systems are connected by their dependence on CRISPR RNA (crRNA). CrRNA helps Cas proteins recognize and cleave nucleic acid targets. Through hybridization to a complementary sequence, researchers can program crRNA toward a specific DNA or RNA region of interest. In some systems, this complementary sequence is restricted to the proximity of an adjacent protospacer motif (PAM) or protospacer flanking sequence.

Researchers have repurposed CRISPR/Cas for several applications, such as the targeted editing of genomes, transcriptomes, and epigenomes; the recording of cellular events; the detection of nucleic acids; and the bioimaging of nucleic acids. Overall, CRISPR-based diagnostics are rapidly evolving, building on CRISPR technologies’ programmability, specificity, and ease of use. Therefore, researchers are coming closer to developing nucleic-acid-based point-of-care (POC) diagnostic tests in routine clinical care.

CRISPR-Based Diagnosis Methods

Since the advent of CRISPR gene editing, many CRISPR/Cas systems have rapidly grown. As a result, there are two classes, six types, and many more CRISPR/Cas systems subtypes. Researchers define these classes, types, and subtypes by the nature of the ribonucleoprotein effector complex.

While multiple effector proteins characterize class 1 systems, class 2 systems incorporate a crRNA-binding protein. Class 2 systems are more commonly utilized in diagnostics as these systems are easier to reconstitute. In addition, these systems include enzymes with collateral activity, which form the backbone of several CRISPR-based diagnostic assays. Researchers have engineered some class 1 systems for diagnostics, either with components of the class 2 system or with the native type three complex.

CRISPR Gene Editing Diagnostics: Cas13

The CRISPR/Cas system’s capacity for precise cleavage of particular nucleic acid sequences grows out of its role as a bacterial “immune system” against viral infections. Because of this link, early adopters of CRISPR gene editing considered the system’s applicability to viral diagnostics. However, all Cas enzymes are different and have different applications.

For example, Cas9 is the ideal enzyme for CRISPR-based genome manipulation. But many CRISPR-based diagnostic approaches make use of Cas13, a family of RNA-targeting molecules that Feng Zhang, a molecular biologist, and his team identified in 2016. Cas13 uses its RNA guide to recognize an RNA target by base-pairing. It then activates a ribonuclease activity that researchers can harness as a diagnostic tool with a reporter RNA. Cas13 doesn’t simply cut the RNA targeted by the guide RNA. It also performs “collateral cleavage” on any nearby RNA molecules.

Many Cas13-based diagnostics use a reporter RNA that tethers a fluorescent tag to a quencher molecule to inhibit fluorescence. When Cas13 recognizes viral RNA and becomes activated, it cuts the reporter and releases the fluorescent tag from the quencher. This generates a detectable signal. Some viruses leave a signature so strong that researchers can achieve detection without performing an amplification step, which simplifies point-of-care diagnostics. For example, Jennifer Doudna and Melanie Ott, from the Gladstone Institute of Virology in San Francisco, demonstrated a rapid, nasal-swap-based, amplification-free CRISPR/Cas13 test for SARS-CoV-2 using a mobile phone camera.

RNA-amplification procedures can improve sensitivity for trace viral sequences. Pardis Sabet, a geneticist at the Broad Institute of MIT and Harvard, worked with her team to develop a microfluidic system that screens for various pathogens at the same time using amplified genetic material from a few microliters of sample. As a result, the research team has developed CRISPR-based detection tools that simultaneously detect over 169 human viruses.

Researchers can use other Cas enzymes in their diagnostic toolboxes too. For example, Cas12 proteins exhibit similar properties to Cas13 but target DNA instead of RNA. As a result, these proteins can detect a broader range of pathogens and enable the effective diagnosis of non-infectious diseases.

BioTechniques’ focus on methodologies advances life sciences research.

When BioTechniques launched in 1983, it was the first publication to focus on lab methodologies instead of the effectiveness of new drugs. The revolutionary journal delved into an essential aspect of the life sciences, as techniques and their repeatability are crucial to scientific and medical advancements yet are often overlooked. Over the years, BioTechniques has published extensive coverage of methods like chromatography, western blotting, polymerase chain reaction, next-generation sequencing, and CRISPR gene editing, both in its print journal and on its multimedia website.