The Potential for Groundbreaking Discoveries With CRISPR Screening

Since its discovery over 30 years ago, the potential therapeutic and diagnostic applications of the CRISPR-Cas system have continued to evolve. Found only in archaea and some bacteria – two of the three domains of life – the CRISPR-Cas system functions as a sort of adaptive immunity for prokaryotes, which, unlike vertebrate adaptive immunity, is heritable.

The system’s heritability to daughter cells is encoded in genomic DNA, which is one of the characteristics of CRISPR that makes it so useful in clinical and translational research. Additionally, CRISPR libraries can be constructed in essentially any manner suitable to the desired experimental outcomes (loss or gain of function) or settings: DNA-based vectors, RNAs or ribonucleoproteins (RNPs).

Upstream of coding sequences for Cas proteins lies a 21–40 bp repeat motif interspersed by 20–58 bp spacer elements, which are integrated sequences derived from invading bacteriophages. The entire region is transcribed, translated and processed so that the CRISPR-Cas RNA complex (crRNA) targets the complementary sequence of subsequently invading bacteriophages. The contiguous trans-activating RNA (tracrRNA) facilitates the nuclease function of the Cas enzyme, resulting in double-strand breaks at the target site. While eukaryotes do possess endogenous RNA systems to modulate gene expression, to date, evidence is still limited on a system similar to CRISPR in eukaryotes.

Taken together, the utilization of CRISPR-Cas has become an extraordinarily powerful tool in clinical and translational research for potential drug therapy targets, functional studies and as a therapeutic itself. Here, we consider various platforms to modulate gene expression, experimental design, current challenges and the potential of CRISPR-Cas systems to understand and treat diseases, from cancers and congenital disorders to overcoming the growing prevalence of antimicrobial resistance.

Platforms for modulating gene expression

Several tools exist for researchers to consider in their experimental designs, including RNA interference (RNAi) using small interfering RNAs (siRNA) derived from eukaryotic genomes, transfection of cells with CRISPR-Cas9 cassettes integrated into plasmid or viral vectors and, more recently, the development of chemically synthesized CRISPR guide RNAs (gRNAs) or CRISPR RNPs directly delivered into cells. Each method has its advantages and drawbacks to consider.

Modulation of eukaryotic gene expression with RNAi has been employed for over 20 years. The underlying premise of RNAi is the use of double-stranded RNA, which forms short hairpin loops that are cleaved by the Dicer enzyme and can then evade mammalian immune responses to selectively knock down target gene expression by interfering with the translation of mRNA into protein.

Because the effects are temporary, researchers studying essential genes – those that are required for cell survival – can modulate the expression of these genes without killing the cells. However, temporary expression may be a drawback in some scenarios in which one wishes to study the long-term effects of modulated gene expression.

In contrast, gene knock-out using CRISPR-Cas is permanent and heritable to daughter cells, although gene activation or inhibition using CRISPR is not. Because the CRISPR-Cas elements are not universally present in bacteria and not at all found in eukaryotes, cells must be provided with an exogenous source.

Sources may include plasmid or virus vector libraries encoding the necessary elements to be integrated into the genome, gRNAs and mRNAs delivered via encapsulation, or the CRISPR gRNA and Cas enzyme delivered as RNPs. While pooled plasmid or viral screening has the benefit of facilitating high-throughput functional screening of genes, it requires host cell transcription machinery to express the vector target sequences and gRNAs.

Furthermore, it can be challenging to determine which of the guide sequences had the desired effect. Determining this requires further downstream sequencing experiments, adding time and cost to the study. Off-target edits may also pose a challenge with less specific gRNAs, not to mention the possible unintended consequences of incidental integration of vector sequences into the genome of interest.

Conversely, arrayed chemically synthesized CRISPR screening libraries do not require a vector for expression within cells; the synthetic gRNAs provided do not require any further transcription or translation and are immediately functional upon transfection and translocation into the cell nucleus. Researchers may transfect cells already capable of expressing Cas9 or may deliver the gRNA/Cas9 as an RNP that can directly target the gene sequence of interest. Such direct delivery has a distinct advantage in experiments using post-mitotic (nondividing) cells, such as neurons.

Arrayed CRISPR libraries can have a higher upfront cost, as they require a unique gRNA for each target and treatment, typically in 96- or 384-well plates. More costly equipment such as robotics is typically utilized in arrayed screening approaches as well. On the other hand, arrayed CRISPR libraries do not require dividing cells or host cell transcription machinery for expression and eliminate the potential for random integration, which can lead to off-target effects. They also eliminate or reduce the need for downstream next-generation sequencing (NGS) to determine disrupted sequences and reduce overall experimentation time. Arrayed gRNA also allows for the use of any subset of the library in downstream experiments rather than needing to build an entirely new library, as is the case with plasmid-based methods.

Experimental design considerations

Important variables for researchers to consider when designing experiments using CRISPR screening libraries include:

• Whether a pooled or an arrayed gRNA library is most appropriate. If gene discovery is the experimental endpoint, a pooled library may be an appropriate choice.
• Whether the desired effect is the complete knock-out of gene expression, retaining some level of expression (inhibition), or activation or overexpression. Choosing which Cas enzyme will facilitate the desired outcome is equally important as the gRNA library.
• For knock-out libraries, whether to use a whole genome library (hypothesis-free) or focused library.
• Choosing which cell line or animal model will be the most informative.
• Deciding which methods should be used to measure the outcome.
• How the desired results will be confirmed. An arrayed screen may be desirable to confirm the results of pooled screens of a large number of genes, such as those potentially conferring drug sensitivity or resistance.

In these considerations, including the appropriate positive and/or negative controls in an experiment will improve the likelihood of correctly attributing cause and effect. While knock-out or inhibition/repression of gene expression is the most common aim for CRISPR gene targets, scientists should also be aware of functional redundancy and whether the gene(s) of interest will be subject to loss of function effects.

The challenges – and potential – for therapeutics

Using CRISPR in an in vivo model system holds great promise for therapeutic applications given its ability to directly manipulate a genome without the risks that viral vectors can pose by integration. Almost 60 clinical trials in various stages of recruitment or completion are listed using “CRISPR” as a keyword in a clinical trial database. These researchers are investigating the potential this technology may have to treat or cure diseases including cancer, infectious diseases and inherited disorders.

Additionally, the ability to generate animal models of disease using CRISPR technology has rapidly changed the early pre-clinical testing phase of drug discovery by facilitating easier gene targeting. 

Recently, ex vivo gene editing using CRISPR technology followed by transplantation was credited for curing a patient of sickle cell disease, a potentially life-threatening condition that affects millions of people worldwide. But not all tissue types are as accessible for targeted transplantation or implantation as bone marrow. What’s more, multicellular organisms are highly complex in terms of spatiotemporal regulation of genes. Delivery systems for less accessible tissues, such as brain tissue, are still needed and research in this area is ongoing.

While the production of edited cells ex vivo, at scale and under Good Manufacturing Practices suitable for human therapeutics remains a challenge, the potential for evolutionarily-conserved CRISPR-Cas as a therapeutic itself – in addition to being a valuable tool for functional studies and drug target discovery – makes for a promising future indeed.

About the author:

Mollie Schubert is innovation product manager – Genomic Medicine & CRISPR at Integrated DNA Technologies, a global genomics solutions provider whose mission is to accelerate the pace of genomics. She has more than 10 years of experience with high-throughput screening of CRISPR-Cas9 guides for the development of a site selection tool, optimizing the composition and delivery of synthetic RNA reagents complexed to recombinant CRISPR nucleases, and developing methods for efficient gene editing. 

Disclaimer: For research use only. Not for use in diagnostic procedures.