Researchers at New York University and the New York Genome Center have developed chemically modified guide RNAs that enhance CRISPR-Cas13 transcript knockdown in human cells. These systems will allow transcripts to be modulated without genetic manipulation, with implications for future disease treatments.
Recently, RNA-targeting CRISPR-Cas13 proteins have emerged as a powerful platform to modulate gene expression. In previous studies, the CRISPR-Cas13 enzyme family has shown remarkable versatility. This has led to suggestions that CRISPR-based transcriptome modulation could hold therapeutic potential for a wide spectrum of RNA-mediated diseases.
The process works through a CRISPR RNA (crRNA) guiding Cas13 to its target by RNA-RNA hybridisation of a short spacer sequence to the target site. However, endogenous RNA nucleases rapidly degrade crRNA in human cells, leading to knockdown being very transient. This makes protein and crRNA delivery an incredibly challenging process. It is also difficult to manipulate the transcriptome without modifying the host’s DNA sequence. The researchers aimed to discover if chemically modifying synthetic crRNAs could overcome these problems.
Alejandro Méndez-Mancilla, co-first author of the study, said: “CRISPR RNA guide delivery can be challenging, with knockdown time limited due to rapid guide degradation. We were inspired by the guide modifications developed for other DNA-targeting CRISPRs and wanted to test if chemically modified guides could improve knockdown time for RNA-targeting CRISPR-Cas13 in human cells.”
Optimal crRNA modifications
The team screened multiple chemical modifications of crRNA. From this, they identified specific modifications that improved RNA targeting efficiency and half-life in human cells.
They found that modifications at the 3’ end of synthesised crRNAs boosted transcript knockdown compared to unmodified crRNAs, especially when modifications were placed within the spacer sequence. 2’-O-methylation at bases on the 3’ end were especially successful. Conversely, modifications at the 5’ end, whether alone or in conjunction with 3’ changes, did not improve knockdown efficiency. Through this work, optimal crRNA modifications were identified, which have the potential to be utilised in future studies.
In addition, the study found that co-delivery of modified crRNAs and recombinant Cas13 enzymes in ribonucleoprotein (RNP) complexes can alter gene expression in human T cells. Modified crRNAs were able to knockdown CD46 expression in the cells by 60%-65%, whereas unmodified crRNAs only achieved 40%-45% knockdown. It is thought that the RNP complex protects the crRNA from degradation, therefore leading to greater knockdown efficiency.
Recently, it has been suggested that Cas13 could be used to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the RNA virus behind the COVID-19 pandemic. Therefore, the researchers designed synthetic crRNA constructs that targeted the leader sequence of SARS-CoV-2. Excitingly, the results revealed that 3’-modified crRNAs can suppress protein expression in SARS-CoV-2. These findings suggest that Cas13, together with modified crRNAs, could represent a potential therapeutic treatment against COVID-19.
The system developed in this study represents a novel and efficient method to modulate transcripts without genetic manipulation. CRISPR modifications have historically been challenging in human primary cells, but the methods used here show that it is possible. In addition, the targeting of SARS-CoV-2 demonstrated the utility of transcriptome engineering, which has huge potential for future therapeutic treatments. Hans-Hermann Wessels, co-first author of the study, said: “We hope the improved effectiveness and stability from these modified CRISPR Cas13 guides will help pave the way for use of RNA-targeting CRISPRs in primary cells.”
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