Have you been overwhelmed by the number of CRISPR articles published in 2017? PubMed alone has cited over 3,000 CRISPR publications in 2017! We wanted to save you from having to sieve through the databases by asking our experts to select what they thought were the most important CRISPR publications from last year.
Below is our list of publications that we reckon have the greatest potential to solve basic biological and drug discovery problems and which will, hopefully, inspire you to explore further. Here’s to ‘Dreaming in a pragmatic way’ for 2018.
1. Improving cancer immunotherapies
A publication by Burr et al., uncovered a protein, CMTM6, which controls the cell surface expression and recycling of programmed death-1 (PD-1) ligand 1 (PD-L1). Using a genome-wide CRISPR screen, the authors found that CMTM6 is required for effective PD-L1 expression in tumour cells and increases their capacity to evade destruction by T cells through stimulation of PD-1 expressed by activated immune cells. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested in this study and in primary human dendritic cells and increases the anti-cancer immune response in vitro and in vivo.
Targeting this previously unrecognized master regulator of PD-L1 expression has the potential to improve the response of human tumours to cancer immunotherapies.
2. Multiplex recording of cellular events over time using CRISPR biological tape
Our capacity to fully appreciate the dynamic nature of biological processes is one with which we are still struggling. Coming to the rescue, Sheth et al., describe their innovative ‘biological tape recorder’, which enables the recording and storage of temporal biological information directly into the genomes of a cell population.
The researchers studied biological signals which triggered intracellular DNA production that was then recorded by a CRISPR-Cas adaptation system. This remarkable approach enables stable recording over several days and facilitates a reconstruction of time sequences and lineage information revealed by sequencing the resulting CRISPR arrays.
The researchers used their biological tape recorder to concurrently record over time the availability of three metabolites (copper, trehalose and fucose) in the environment of a cell population.
Why do we think that this work is significant? We anticipate that the insight gained from this ability to measure the dynamic cellular states and environmental changes over set time periods will enable a better understanding of a cell’s natural rhythm in defined environments. This might reveal in more detail, specific vulnerabilities of cells to particular types of drugs, for example. In addition, this paper has already resulted in suggestions for chronicling biological events on a large scale.
3. Dual-CRISPR screening finally reveals a drugs elusive mechanism of action
Jost et al., use the parallel application of both CRISPRa and CRISPRi to solve a decades-old riddle — what is the mechanism by which Rigosertib exerts its effect? Rigosertib was identified as a potential anti-cancer drug through a phenotypic screen, and although now in Phase III clinical trials, its MOA was still undefined.
By taking a genomics approach and examining the function of the drug in both loss and gain-of-function analyses, the authors uncovered the unique genetic signature of the drug response. These data unambiguously revealed that Rigosertib is a microtubule-destabilising agent. This paper indicates that CRISPRi and CRISPRa screens when used in combination have phenomenal power to provide robust insight into a drugs MOA. Moreover, this paper sets a new gold standard for drug-gene interaction studies.
4. Identifying synthetic lethal drug target pairs
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but how can you carry out direct screening of all possible drug combinations? Overcoming this problem, Han et al., used a powerful CRISPR-based double knockout (CDKO) approach to improve the efficiency of combination genetic screening. The authors used an effective strategy for cloning and sequencing paired single-guide RNA libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs.
The researchers then applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against over 20,000 pairs of drug targets in leukaemia cells and identified synthetic lethal drug target pairs for which corresponding drugs demonstrated synergistic killing.
This research lays the ground work for future strategies to screen for synergistic drug combinations at high-throughput at the genetic level. It also showcases how a CRISPR-based tool can be used to dissect functional GI networks.
5. Using the “Big Papi” to study phenotypes
Like the Han et al., publication highlighted above, uncovering redundant genes and exploring complex gene networks can be done by combination genetic ablation screening using pooled CRISPR-Cas9 libraries. However, there’s a catch — these methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity.
To mitigate these problems and increase the efficiency of combinatorial screening, Najm et al., used orthogonal Cas9 enzymes from S. aureus and S. pyogenes. These authors first established S. aureus Cas9 sgRNA design rules using machine learning approaches and then paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual gene targeting in a large proportion of transduced cells. As a proof of concept, they applied this orthologous approach, which they termed “Big Papi” (short for Paired Aureus and Pyogenes for Interactions), to screen for synthetic lethal and buffering gene interactions and found both known and novel combinations of paired genes that affected cell viability.
Interestingly, these authors also used “Big Papi” to enable a dual screen in which gene knockout is combined with transcriptional activation. This approach revealed genetic interactions that buffer the lethality induced in cancer cell lines through the overexpression of wild type p53. Although still confined to the small-scale focused interrogation of a few hundred genes, this technology is widely applicable to the study of combinatorial phenotypes and holds great promise for uncovering novel genetic interactions in most biological processes of interest.
New ethical considerations and conclusions
2017 also saw several papers demonstrating the use of CRISPR in viable human embryos. CRISPR was employed to help understand basic reproductive and developmental biology, correct heart defects and carry out mitochondrial replacement therapy, to name but a few.
The ethics of creating designer babies and the worry of possible off-target mutations raises a number of questions on the correct translational use of CRISPR. As the explosion of CRISPR publications will no doubt continue in 2018, its refinement in technical and ethical terms will also occur.