Introduction

Although much of the reporting around CRISPR tends to focus on its cleavage based ability to knock out or repair targeted sequences of DNA in a genome, particularly for human therapeutic or agricultural applications, some of the most exciting developments in the CRISPR field do not involve gene editing at all. Many of these advances are already being put into effect in basic science research, much sooner than any CRISPR-based treatment or agricultural product will likely become available. The broad utility and impact that these new CRISPR-based research tools may have not only on basic science, but also molecular diagnostics and drug discovery and research may in fact come to rival the oft hailed potential of the system as used to directly create human therapeutic and agricultural products.

CRISPR base editing and gene expression modulation

Scientists around the world are coming up with ever more ingenious ways of using the flexibility and sensitivity of the CRISPR system (whose ease of use is its key differentiator compared to earlier ZFN or TALENs based technology) to devise new variations of the system with new and different functional applications. We previously reported on 25 October 2017 and 23 September 2017 about the “base editor” variations of the CRISPR system which use dead Cas9 or a Cas9 nickase linked to other enzymes to effect single base changes around the target site. CRISPR-based modulation of gene expression, through the combination of a sgRNA-dcas9 targeting component with transcriptional repressors, activators or epigenetic modulators is also being explored.

In a paper from the Whitehead Institute just published in Cell, dCas9 was fused to demethylation enzyme Tet1 in order to effect the targeted demethylation of the CGG expansion region upstream of the 5’ UTR of the FMR1 gene, which is silenced in Fragile X Syndrome (the most common genetic form of intellectual disability in males). Hypermethylation of CGG repeats in that region is thought to be associated with the silencing of the gene. Through this targeted demethylation, the researchers were able to restore the expression of FMR1, in neurons derived from induced pluripotent stem cells, to close to wild type levels, which effectively reversed the abnormal electrical activity symptomatic of Fragile X and restored the wild type phenotype. When those modified neurons were engrafted into a mouse brain it was found that the FMR1 gene remained active for at least three months. There were some off-target effects, but these were found to be low and able to be reduced by using less of dCas9-Tet1.

The base editor and gene expression modulation variations of the CRISPR system have sometimes been dubbed “CRISPR 2.0” in the popular press. The common theme of these new variants is their utilisation of the essential sgRNA-based targeting component of Cas9 (as opposed to its cleavage activity) in order to specifically target a region of DNA for the delivery of a separate, different protein to achieve the desired effect. This is an incredibly flexible and powerful tool – in theory one could link almost anything to the sgRNA-dCas9 complex, which will then home in on the target sequence complementary to the sgRNA and deliver its payload.

CRISPR based molecular diagnostics and biosensor applications

One of the key emerging applications which takes advantage of the sgRNA mediated targeting ability of CRISPR is to sensitively detect the presence (or absence) of particular target molecules, or cellular events. Sensitive and reliable molecular diagnostics (whether as a companion diagnostic or more generally) is a key pillar of the precision medicine revolution – the ability to accurately detect small amounts of circulating tumour DNA, viral DNA or cell free fetal DNA (to name just a few applications) may allow for early diagnosis and/or preventative treatment, in addition to enabling the selection of an appropriate targeted treatment based on the genotype of the patient and/or disease-associated entity.

DETECTR, CAMERA and SHERLOCK are the acronyms that researchers have given some of the latest of these diagnostic manifestations of CRISPR technology, which are being developed by some of the leading lights in the field. DETECTR, from Jennifer Doudna’s University of Berkeley lab and SHERLOCK, from Feng Zhang’s lab at the Broad Institute are both techniques designed to detect attomolar quantities of target DNA (e.g. viral DNA present in the blood of a patient). Meanwhile CAMERA has been developed by David Liu at the Broad as a technique that allows the recording of the occurrence of particular cellular events (e.g. exposure to particular stimuli, or the activation or repression of certain coding regions or signalling pathways).

Detecting DNA with CRISPR

At a high level SHERLOCK and DETECTR both operate by way of the same basic theory:

1. A “recombinase polymerase amplification” (RPA) system is used to amplify low copy numbers of the target DNA sequence (to increase the sensitivity of the assay) and generate the substrate for CRISPR mediated binding cleavage. In the case of DETECTR this is the double stranded DNA product of the amplification reaction, but for SHERLOCK a T7 polymerase is used to generate RNA from the amplified DNA.

2. A CRISPR-Cas protein that possesses secondary “collateral” cleavage activity upon binding and cleavage of its primary target is introduced, together with reporter molecules comprising a linked fluorophore and quencher.

3. Upon cleavage of the primary target, the secondary cleavage activity chews up the link in the reporter molecules, freeing the fluorophore and giving rise to a fluorescent signal that indicates the presence of the original target sequence (similar to how probe sequences operate in real time PCR).

DETECTR (DNA Endonuclease TargEted CRISPR Trans Reporter)

The Cas protein variant relied on in the DETECTR system is Cas12a (aka Cpf1), which Doudna’s group discovered has the ability to indiscriminately cleave single stranded DNA after the Cas12a complex has bound to and cleaved its target site in dsDNA. The reporter molecules used in the DETECTR system are therefore linked by a length of ssDNA, which is degraded upon Cas12a binding, to release the fluorophore.

To demonstrate proof of concept, the Doudna lab team used this technique to specifically detect two high risk strains (HPV 16 and HPV18) of human papilloma virus, which causes cervical cancer, at attomolar concentrations from 25 samples infected with many different types of HPV. The presence or absence of HPV16 was correctly identified in all of the samples (8/8 positive, 17/17 negative) and HPV18 was correctly identified in all but two (6/8 positive, 17/17 negative).

The team noted that their finding of this new ssDNA cleavage activity of Cas12a will require further investigation, and may have implications for the use of Cas12a in vivo, where most DNA is double stranded, but occasionally unwound into single strands during various cellular processes such as during transcription. Unsurprisingly, Zhang has downplayed the impact of the finding for the potential safety of Cas12a, stating that the enzyme should be bound to genomic DNA and restricted in its ability to roam around the cell to find ssDNA targets.

It is worth noting that, unlike CRISPR-Cas9, the position in respect of the ownership of the patents covering the CRISPR-Cas12a system is clear – coming out of work done by Feng Zhang’s lab, those patents are owned by the Broad Institute and its collaborators and exclusively licensed to Editas Medicine for human therapeutic applications. Should Doudna or Berkeley seek to commercially develop the DETECTR system then a licence from the Broad and/or Editas would be needed.

SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing)

SHERLOCK, as first reported by Zhang’s group in 2017, used Cas13a (aka C2c2), which binds to and cleaves RNA at the target site complementary to the guide sequence, before cutting other nearby RNA sequences (in this case, RNA based reporter molecules). The most recent iteration of the technique is said to be 100-fold more sensitive than the previously reported version, can be multiplexed using different sets of Cas proteins (including the Cas12a used in DETECTR) and reporter molecules and is able to visualise results on paper strips similar to those used in pregnancy tests.

The multiplexing was achieved by the use of different Cas proteins with collateral activity specific to different nucleotide motifs in the reporter molecule. By pairing different Cas proteins targeting different DNA regions with corresponding reporter molecules with different fluorophores, the presence of multiple different targets in the one sample can be detected. The Cas variants employed for the purposes of multiplexing were LwaCas13a, PsmCas13b, CcaCas13b and AsCas12a.

Zhang’s team tested their improved SHERLOCK system on samples including Dengue and Zika virus as well as circulating tumour DNA, finding that they were able to detect targets down to attomolar concentrations (and even beyond, if the RPA step is scaled up). In various other experiments the researchers also found that by diluting the primer concentration they could achieve a degree of quantitation in the assay, and that by synergistically combining the activities of Cas13 and Csm6, the signal from the assay could be boosted, reducing background and the risk of false positive readouts.

These findings were brought together and incorporated into the design of a system to visualise the results of the assay by way of lateral flow on paper strips, without need for instrumentation. This has been touted as a major development which brings the system closer to potential field use across a wide range of applications and industries, due to its relative portability, ease of use and sensitivity.

CAMERA (CRISPR-mediated Analog Multi-Event Recording Apparatus)

CAMERA is the latest in a series of CRISPR-based “cellular recorders” which seek to allow researchers to keep track of cellular events such as changes in gene expression or exposure to particular environmental conditions. The basic theory behind the technique is to tie expression of the Cas9 protein and/or sgRNA sequence to a particular stimulus to be recorded. When the cell experiences that stimulus (in this case, exposure to a specific antibiotic), the Cas9/sgRNA complex is produced and proceeds to effect its recording function.

In the first variant of the system (CAMERA1), the researchers employed a mixture of wild type and modified plasmids as a recording device. The modified plasmids were engineered to contain a target region for the Cas9/sgRNA complex, such that when those components are expressed in response to the stimulus to be recorded, the modified plasmid would be degraded and replaced with a wild type plasmid. The amplitude and duration of the stimulus can thus be observed by analysing the ratio of modified to wild type plasmids.

In the second variant of the system (CAMERA2), the team used base editors (which Liu first developed in 2016) as the recording mechanism. These base editors performed single base edits directly in the genome of the cell, with different edits associated with different stimuli, thus allowing for multiplex recording of various stimuli in the same cell. The amplitude and duration of the stimulus can then be assessed by reference to the proportion of DNA in which each single base change has been made.

Conclusion

As the toolbox of application to which CRISPR based systems have been applied continues to expand still further, it seems that the sheer range of different uses to which CRISPR can be put is only limited by the imagination and ingenuity of researchers. In a mere few years, the sheer versatility and power of the system has already revolutionised basic science research and looks set to do the same for diagnostics and therapeutics. As these technologies develop and the precision medicine field continues to mature, CRISPR seems sure to play a leading role every step of the way – from basic research and targeted drug discovery, through to diagnostics and ultimately treatments for genetic disease.