The trailer of the new Netflix docuseries Unnatural Selection, released in early October, proclaims, “From eradicating disease to selecting a child’s traits, gene editing gives humans the chance to hack biology.” The series centers around genetic engineering and, in particular, CRISPR from the perspective of scientists, companies, and biohackers. The show’s cocreator, Joe Egender, has said, “Our main hope is to create a discussion around these technologies. People might come away excited. Or they might be scared. But at least that means they are talking and learning and understanding what’s coming.” The show’s release promises to bring CRISPR gene-editing tools to the forefront of mainstream discussions.
The CRISPR-Cas system is an RNA-guided nuclease that has been adapted to cleave DNA or RNA at desired locations within a genome. It has already been touted as one of the most significant discoveries of the 21st century. We have previously explained the general concept and mechanisms of the CRISPR-Cas system and introduced potential CRISPR applications on the horizon. Here, we detail some of the top CRISPR breakthroughs of 2019, which can help us to understand current advances and anticipate the future of this technology.
CRISPR for treating HIV:
CCR5 is a coreceptor of HIV, via which the virus targets CD4 T cells to suppress their immune response. It has already been demonstrated that CCR5 mutations that lead to nonfunctional proteins could make cells resistant to HIV. Chinese scientist Hongkui Deng and coworkers have started to use the CRISPR-Cas9 system to render the CCR5 gene nonfunctional in stem cells and they have transplanted these stem cells into an HIV patient diagnosed two years prior. Recently, they published a case report in the New England Journal of Medicine revealing that although the patient’s HIV infection was not cured, no unintended genetic alterations were detected, indicating that the therapy is likely safe.
The work Hongkui Deng and his team have done is ethically different from what Jiankui He did last year. Of note, Jiankui He used CRISPR to edit the CCR5 gene in human embryos, attempting to make unborn twins immune to HIV. However, any off-target genome edits could have lead to detrimental effects in the babies. This is in stark contrast to Deng’s study using CRISPR to edit CCR5 in adult stem cells that were then transplanted into a patient, as the CCR5 gene would remain functional in non-blood cells.
Researchers make record-breaking 13,000 edits in a single cell by CRISPR:
A study led by George Church from MIT succeeded in making 13,000 edits by CRISPR in a single cell, which is approximately 2,000-fold higher than that achieved by the same group collaborating with Luhan Yang’s group in 2015, wherein they mutated every single PERV gene in a porcine primary cell line. PERVs are viruses that incorporate their genetic materials into pig genomes. In this new study, the researchers designed a set of dead-Cas9 base editor (dBEs) variants that enable the editing of thousands of loci in each cell without causing cell death (too much DNA cleavage always leads to cell death). Their dBEs allow up to 13,200 cleavages in 293T cells.
Prime editing makes CRISPR more powerful and precise:
Base editors, as mentioned above, were initially developed by researchers from David Liu’s group and allow for the direct conversion of a single nucleotide into another without breaking the DNA or RNA in eukaryotic genomes. Importantly, however, base editors cannot delete or insert specific sequences at desired positions.
Recently, researchers from the Liu Lab have developed a new CRISPR editing system called prime editing that enables the deletion and insertion of specific sequences, as well as correction of any type of point mutations in cellular DNA. The prime editor complex is composed of an engineered Cas9 that creates a single nick on one DNA strand, a prime editing guide RNA (or pegRNA, which is comprised of “guide sequences” that guide the whole complex to the target and the edited sequence that is designed to replace the old target sequence), and a reverse transcriptase than can make new DNA by reverse transcription of the edited sequence of pegRNA. Besides its ability to insert and delete specific sequences, prime editing also has enhanced efficiency and accuracy compared to the other engineered Cas9 variants. This study also indicates that prime editing has the potential to correct roughly 89% of human pathogenic variants, which increase susceptibility or predisposition to a certain disease or disorder.
CRISPR-Chip enables highly sensitive digital detection of DNA:
Scientists are now turning the CRISPR technique into a Swiss Army knife by exploring its application in various fields other than gene editing. Recently, a study conducted by researchers from the Keck Graduate Institute and the University of California, Berkeley, used CRISPR-Cas9 and a graphene chip to detect DNA. By combining the two, the conventional amplification step is no longer necessary to detect DNA due to CRISPR’s genome-searching capability and graphene’s high sensitivity. According to their paper published in Nature Biomedical Engineering, CRISPR complexes were immobilized on graphene-based transistors.
When the CRISPR-Cas9 complexes find and bind their target DNA, the conductivity of the graphene material in the transistor changes; this change can be detected by a reader developed by the group’s industry partner, San Diego–based Cardea Bio. The researchers also demonstrated the effectiveness of detecting DNA mutations in Duchenne muscular dystrophy (DMD), a genetic disorder caused by a mutation of the gene that prevents the body from producing dystrophin, resulting in progressive muscle degeneration.
CRISPR-Cas13 is programmed to kill and detect RNA viruses in human cells:
The CRISPR family has been expanding rapidly since the discovery of its ability to edit the human genome. Researchers at Zhang Lab originally revealed that Cas13a could bind and cleave RNA in human cells, which makes it a potential diagnostic tool to detect viruses, bacteria, or other targets.
Recently, scientists from Harvard and MIT’s Broad Institute explored CAS13’s ability to detect and kill RNA viruses, many of which are detrimental and incurable human pathogens such as Ebola and Zika. The researchers combined Cas13’s antiviral activity with its diagnostic capability to create a system called Cas13-Assisted Restriction of Viral Expression and Readout (CARVER) to detect and kill RNA viruses. The scientists demonstrated the antiviral efficiency of CRISPR-Cas13 in human cells infected with lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), or vesicular stomatitis virus (VSV). They also incorporated the Cas13-based nucleic acid detection technology SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) to enable the system to serve as a “detective” as well.
Targeted cancer drugs may miss their targets:
Targeted cancer drugs, which are designed to specifically target tumor-related proteins to suppress tumor growth and progression, have fewer side effects compared to traditional cytotoxic therapies and have shown promise in some cancer treatments. However, a recent study found that many targeted cancer drugs may miss their target proteins. The researchers from Jason Sheltzer’s group used CRISPR-Cas9 to eliminate the genes of the presumed target proteins of these targeted cancer drugs, but the drugs still effectively killed cancer cells. This indicates that the interpretation of the mechanisms of how these cancer drugs function on cancer cells is possibly inaccurate, and they may thwart cancer cells by other mechanisms rather than directly targeting the presumed target proteins.
This is not surprising at all, as some rationally designed targeted anticancer molecules do not kill cancer cells. The CRISPR technique can help us better understand the pharmacology of such targeted anticancer drugs, thereby leading to more efficiently designed anticancer drugs in the future.
Future efforts:
Though there have been ever more exciting breakthroughs for CRISPR, many obstacles remain to be overcome. One of the biggest obstacles is that we are still not able to control the off-target effects of CRISPR, which would be extremely detrimental if they were to happen in patients. Therefore, more work needs to be done to improve the fidelity and accuracy of the CRISPR technique. In addition, the recognition of the target sequence by most CRISPR-Cas systems still depends on a PAM sequence, which limits its application to a broader range of diseases, as not all genes contain such specific PAM regions in locations of interest. Importantly, aside from these technical limitations, more attention must be given to regulations of CRISPR applications to avoid potential ethical issues or tragedy.
Featured image source: Ernesto del Aguila III, National Human Genome Research Institute, NIH
