Chemically modified guide RNAs to expand CRISPR functionality

CRISPR-Cas, a bacterial adaptive immune system against viruses, has been repurposed into a biological research tool that is invaluable due to its simplicity and programmability. Composed of a Cas endonuclease that is directed to its nucleic acid target by a guide RNA (gRNA), CRISPR has been used for genome editing, live-cell imaging and even diagnostics. In this work, the goal is to chemically modify gRNAs to expand CRISPR functionality. A ‘split-and-click’ strategy for generation of Cas9 gRNAs via CuAAC ‘click’ ligation of shorter oligonucleotides is developed. The approach provides a simple and cost-effective route to individual or pools of single guide RNAs (sgRNAs), while providing scope for further chemical modification to improve sgRNA activity. The click sgRNAs are functional both in vitro and in cells, demonstrating artificial linkages can be tolerated in functionally critical regions of sgRNA. To illustrate the capabilities of the system, work towards an on-field forensic analysis of Short Tandem Repeats (STR) is described using CRISPR, Ramified Rolling Circle Amplification (RAM) and Oxford Nanopore sequencing. sgRNAs were also tagged with small molecule fluorophores to provide greater flexibility in colour choice and expand the colour range for live-cell imaging. Imaging of repetitive elements of telomeres is feasible with these constructs if GFP-tagged dCas9 is used for cross validation; due to high cytoplasmic background fluorescence, methods to turn on fluorescence in the nucleus are also explored. Finally, two CRISPR-AsCas12a gRNAs are chemically ligated to yield a 3'-3' dual gRNA with the aim of altering the DNA cleavage pattern of the Cas12a protein. Chemical ligation in this context impaired gRNA activity in vitro, however unexpected single-stranded DNase activity is described for AsCas12a.