Saturation prime editing

VaLiAnT can be used to generate libraries for Saturation Prime Editing (SPE).

Prime editing works through the coupling of a nickase Cas9 to Reverse Transcriptase domain. A pegRNA, complexes with Cas9 and binds to a gRNA binding site, the pegRNA also contains a Reverse Transcriptase Template (RTT) DNA tract which includes a desired edit to genomic DNA sequence. This template is processed by the Reverse Transcriptase fusion to provide DNA template and the nicked genomic DNA repaired using the locally synthesised homologous RTT sequence.

The pegRNA which consists of distinct DNA segments that have a different role in the prime editing process; a sgRNA binding site spacer, an sgRNA scaffold, a Primer Binding Site (PBS) homologous the spacer sequence and a Reverse Transcriptase Template (RTT). In order to facilitate cloning (into RNA Polymerase III expression plasmids, such as ‘pU6-pegRNA-GG’ – Addgene #132777 (Anzalone et al., 2019)) cloning adapters are added to 5' and 3' of the pegRNA sequence.

For the purposes of pegRNA construction through VaLiAnT the RTT sequence is considered to be a targeton sequence range. Sequence is retrieved based on genomic coordinates entered into an input file and user-defined regions within the RTT targeton subjected to mutator functions to generate variant harbouring sequence.

In order to add 5' and 3' (unedited) sequence segments to construct the full pegRNA VaLiAnT functions adapter-5 and adapter-3 are used:

• the 5' cloning adapters, spacer sequence and scaffold sequence are appended to the RTT targeton using the adapter-5 function.
• the PBS and 3' cloning adapters are appended using the adapter-3 function.
• primer binding sites to allow for generic amplification (such as Illumina P5 and P7 sequences without flowcell binding regions) can be added to distal ends, this is helpful to increase starting material from low yield ssDNA chemical synthesis platforms.

Depending on the orientation of the sgRNA binding site, the RTT is required to be in either the sense or antisense orientation (RTT and sgRNA binding site are required to be in opposing orientations). VaLiAnT allows for this aspect to be included in pegRNA design through the revcomp-minus-strand feature.