Date: 7th January 2020
The ethical debate surrounding the use the CRISPRs is currently at an all-time high. Aside from how they should be used, mitigating off-target effects is one crucial step in the right direction toward improving their safe use. Now scientists from China have achieved conditional control of gene editing in live cells in an attempt to reduce undesired editing.
In 2019, we saw the first patients treated with CRISPR-edited cells and, with the initial short term data looking promising, it is likely that 2020 will see an increase in the number of FDA-approved CRISPR trials starting to recruit and treat patients.
One area of ongoing concern, however, surrounds the potential of unintended edits that can arise from CRISPR-Cas systems. Whilst efforts are being made to develop tools to rapidly catalogue CRISPR unintended edits others are leaning towards improving the CRISPR-Cas system itself.
A constitutively active CRISPR-Cas system is likely to have a relatively high risk of off-target effects. A team led by Xiang Zhou from Wuhan University, China, have therefore introduced post-synthetic masking and chemical activation of guide RNAs (gRNAs) to modulate the cleavage of DNA or RNA by CRISPR-Cas. By conditionally controlling the gRNAs they hoped to reduce Cas activity at sites away from those specifically being targeted to reduce so-called off-target effects.
In order to control and regulate the structure and function of gRNAs the team reasoned that conjugating a chemical moiety to the gRNA would interfere with its interactions with other biomolecules.
Previously published work had shown that introducing a small molecule-removable protecting group – an azidomethylnicotinyl (AMN) group – into a protein of interest could control its function. In this work published by Alexander Deiters and his team in Nature Chemistry, the AMN group was successful engineered into a range of proteins including Cas9, and subsequently blocked interactions with the gRNA to render Cas9 inactive.
‘Masked’ gRNAs
Here, in Zhou’s paper published in Nature Communications, the team covalently attached AMN groups to a variety of gRNAs to create acylated gRNAs. The beauty of this gRNA ‘masking’ method being that it was reversible and the protecting AMN group could be removed by treatment with phosphine via a simple chemical reaction know as a Staudinger reduction.
Initial in vitro work by the group showed that acylation of gRNAs against a reporter gene (green fluorescent protein) efficiently blocked site-specific DNA cleavage by Cas9. To determine the reversibility of the system, de-acylation or ‘unmasking’ of the gRNA was performed using a variety of phosphines. Three phosphines were shown to be particularly effective in the recovery of site-specific DNA cleavage by Cas9 and the gRNA adducts were also shown to be stable under physiological conditions, providing a solid starting point to test the system further.
To do this the group then looked at another Cas family member, Cas13 which is becoming an increasingly important tool, including its recently reported use as a detector and protector in the fight against viral infections. Unlike its Cas9 counterpart which cleaves DNA, however, Cas13 cleaves RNA. To determine whether ‘cloaking’ the crRNA (crispr RNA, part of the gRNA that sequence is complementary to the target site) would render Cas13 inactive, the system was therefore tested against fluorescently labelled target RNAs. Once again, significant masking-dependent inhibition of cleavage was observed, which was reversible in both a concentration- and time-dependent manner following treatment with phospines.
Having successfully demonstrated the application of this method for both Cas9 and Cas13, the next proof-of-concept work was to translate the method into living cells. Using a human cell line expressing Cas9, the team chose endogenous HBEGF (heparin binding EGF like growth factor) and ANTXR1 (Anthrax toxin receptor 1) as target genes. The team showed in-cell CRISPR function was inhibited by chemical masking of gRNA, and that uncloaking could be achieved by treatment with phospines, which reactivated Cas9 target gene cleavage. Furthermore, when compared to a previously published method using gRNA conjugated to photocleavable protectors, the method described here reportedly allowed more controllable gene editing in the cells.
The authors concluded that “Although our strategy is yet to be tested in animal models, the robustness of this strategy and the biocompatibility of Staudinger reduction underline its therapeutic potential”
Future applications
The strategy here of acylating gRNA is an exciting step forward in the temporal control of gene editing by CRISPR systems. Conditional control in this way should in theory reduce off-target effects, and in fact the authors did show that chemically ‘unmasked’ CRISPR systems retain a similar efficiency of on-target cleavage whilst also showing reduced off-target activity, at least in vitro.
Of course, a more thorough investigation into off-target effects both in vitro and more crucially in vivo will need to be determined before this method is accepted as a go-to tool.
The next step for this ‘masking’ tech must lie in extensive in vivo testing. As a point of interest the chemical masking here also provided protection of the gRNAs from RNase cleavage, to which they are typically susceptible in vivo.
It should be noted, however, that this method is not currently compatible with ribonucleoprotein (RNP) complexes. The RNP method can often be used in cells that are difficult to transfect, and it can alleviate difficulties with protein expression. As foreign DNA is not inserted, and the Cas9-gRNA RNP is degraded over time this can also reduce off-target cleavage.
We have previously reported RNP’s encased in nanocapsules or nanoparticles that deliver Cas9 and their gRNAs into the nucleus. Such encapsulation can be used to increase extra-cellular stability and allow targeting to specific cells. Whilst the authors see the acylated CRISPR system as an robust alternative to CRISPR-RNPs it is likely that nano-delivery systems may prove to be compatible or adaptable to this technology and we will awaiting developments on this front.
However, whilst still at the early stages the masking technique reported here could offer an exciting method of controlling gene editing. As a proof-of-concept technology, it is versatile, and rapidly adapted to a given target gene and it presents the synthetic biologist with an additional resource for genome manipulation. Indeed, with increasing numbers of tools now becoming available to determine off-target genome edits we continue to aspire to higher levels of stringency in our quest for novel therapeutic applications.
Wang, S.-R., L.-Y. Wu, H.-Y. Huang, W. Xiong, J. Liu, L. Wei, P. Yin, T. Tian and X. Zhou (2020). “Conditional control of RNA-guided nucleic acid cleavage and gene editing.” Nature Communications 11(1): 91.
https://doi.org/10.1038/s41467-019-13765-3
