2020 was a big year for CRISPR. Jennifer Doudna and Emmanuelle Charpentier shared the Nobel Prize in Chemistry for their pioneering work in the development of CRISPR, the genome editing tool that is leading to revolutionary applications in medicine, agriculture, and basic research.
We previously explained the general concept and mechanisms of the CRISPR-Cas system, introduced potential CRISPR applications on the horizon, and detailed some of the top CRISPR breakthroughs of 2019. Here, we summarize the CRISPR breakthroughs of 2020 in basic research, including diagnostic application, new engineered CRISPR-Cas variants, base editors, and delivery systems. Keeping up to date with these breakthroughs can help us understand current advances and anticipate the future of this technology.
CRISPR progress in diagnosis:
2020 was full of surprises, with the global pandemic of COVID-19 being possibly the most detrimental. This experience has taught us that high-throughput, fast, low-cost diagnostics are key to tracking and controlling emerging global infections.
To meet these requirements, researchers at the Broad Institute of MIT and Harvard developed a testing platform called CARMEN (Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids). It is an extension of SHERLOCK (Specific High-sensitivity Enzymatic Reporter un-LOCKing), a technique for nucleic acid detection with CRISPR nucleases. CARMEN adds the capability of simultaneous detection of multiple nucleic-acid targets, making it a high-throughput method.
The combination of CARMEN and the RNA targeting enzyme Cas13 (CARMEN–Cas13) enables robust testing of infections in a sample, including SARS-CoV-2, the virus that causes COVID-19, via readout of a fluorescent signal. While this method is not yet mainstream, it provides an alternative high-throughput method to detect future infections.
New engineered CRISPR-Cas9 variants recognizing broader PAM range:
CRISPR-Cas9 requires a specific protospacer-adjacent motif (PAM) next to the target sequence to recognize that target, which sets a restriction for more widely sequenced accessibility. In the past year, several research groups have expanded upon the PAM range by engineering Cas9. Another group from the Broad Institute identified three new SpCas9 variants that recognize novel PAM sequences using the continuous evolution method. A group from MIT exploited the novel PAM-interacting loop of a Streptococcus canis Cas9 (ScCas9) ortholog to engineer a new Cas9 that further broadens the PAM range.
New CRISPR base editors:
Existing adenine and cytosine base editors only enable A-to-G or C-to-T transitions. Two research groups have independently developed base editors that combine both cytosine and adenine base editing functions using different methods.
One group from Japan fused the cytosine deaminase PmCDA1, the adenosine deaminase TadA, and a Cas9 nickase (Target-ACEmax) to enable the complex to edit C-to-T and A-to-G simultaneously.
Another research group in the United States developed a smaller dual-base editor by engineering a single protein harboring both adenosine and cytidine deaminases and fused it with the SpCas9 nickase. This smaller-sized base editor makes it easier to be delivered. The group also developed base editors that can efficiently convert base C to G. C-to-G editing enables the introduction of new codons and sequences at the target site, which could potentially permit corrections of more mutations that cause diseases and expand the application of CRISPR and base editing.
Progresses in CRISPR delivery systems:
In the past year, several research groups have made progress in developing or modifying CRISPR delivery systems, advancing the therapeutic translation of CRISPR-based treatments.
Researchers in Israel developed an improved lipid nanoparticle (LNP) delivery system that can more safely and efficiently deliver Cas9 RNAs to tumor targets. Their results show that, with the delivery system they developed, CRISPR can edit up to 70% of the target gene with a single intracerebral injection into aggressive orthotopic glioblastoma in mice. This induces the tumor cells to undergo apoptosis, thereby inhibiting tumor growth. They also engineered their delivery system for antibody-targeted delivery, which results in selective uptake into ovarian tumors and enables up to 80% gene editing of the target gene.
Genome editing by CRISPR-Cas systems is not only about cleavage, but also about knocking in some genes. A collaboration of several labs developed a delivery system by combining supramolecular nanoparticle (SMNP) and supramolecular nanosubstrate-mediated delivery (SNSMD) to deliver CRISPR-Cas9 and knock the hemoglobin beta (HBB) gene into the designated site.
Another collaboration developed a system that can co-deliver CRISPR-Cas9 ribonucleoprotein (RNP) and the antitumor drug photosensitizer chlorin e6 (Ce6) and control the release of the RNP in the tumor cell cytoplasm. They used near-infrared (NIR) and reducing agent–responsive nanoparticles to create this delivery system.
With NIR irradiation, the Ce6 encapsulated in the nanoparticles produces reactive oxygen species (ROS), which causes the release of the nanoparticles from lysosome into cytoplasm, where the RNPs are released and can then locate and modify the target gene to increase the tumor sensitivity to ROS. When NIR irradiation is not applied, the Cas9 will be degraded in the lysosome, preventing CRISPR-Cas9 gene editing in normal cells.
Finally, researchers from UCLA developed a DNA nanoclew–based system to deliver CRISPR-Cas12a for cholesterol regulation. Their DNA nanoclew can efficiently load the Cas12a/crRNA RNP. After being engulfed by the endosome (acidic environment), the DNA nanoclew loaded with RNPs became positively charged and then located efficiently to the target sites for editing.
Future directions:
Though great progress has been made, CRISPR-Cas systems are far from refined. Substantial efforts, including engineering Cas proteins as well as single guide RNAs (sgRNAs), have been made in the past years to reduce off-target effects, but many of them have shown low on-target activity. Therefore, more efforts are needed to reduce off-target cleavage while maintaining robust on-target activity before these systems are widely translated into therapies.
In the past year, many base editors have been successfully developed that can precisely make base transversions. Continuous optimization of such base editors is needed before they advance into clinical applications.
Lastly, though CRISPR-Cas and base or prime editors themselves can be further optimized, efficient and safe delivery of the editing system to target sites remains an essential step for clinical applications, Progress has been made toward enhanced delivery systems, but few have been clinically tested.
Continued development along each of these fronts will further refine our gene editing capabilities and improve upon delivery mechanisms — essential steps in translating CRISPR’s therapeutic capabilities into the clinic.
