Improved prime editing allows for routine predictable gene editing in Physcomitrium patens

Abstract Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3ʹ extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.


Supplementary Data
Table S1: Sequences of pegRNAs used for PpAPT and PpDEK1 Prime Editing Table S2: 3' epegRNA extension sequences Table S3: Primers used in this study Table S4: Prime Editing efficiency and quality of pegAPT#3 variants using fused and split PE systems in P. patens.S3).n: number of edited plants that were sequenced.

Fig. S1 :
Fig. S1: Maps of the expression vectors used in this study.

Fig. S3 :
Fig. S3: Prime Editing strategy for the precise modifications of PpAPT and PpDEK1 genes.

Fig. S8 :
Fig. S8: Examples of sequenced predicted off-targets locus of apt mutant plants using Prime Editing with epegAPT#2.

Fig. S3 .
Fig. S3.Prime Editing strategy for the precise modifications of PpAPT and PpDEK1 genes.(A) Structure of the PpAPT and PpDEK1 genes and pegRNAs positions.Boxes correspond to exon, line to intron.Black boxes define the gene open reading frames.The pegRNAs positions are indicated in red and primers used for PCR and sequencing in green.(B) pegRNAs expected edit information at the two tested loci, PpAPT and PpDEK1.The type and nature of mutations, position on RT product, length of RT template and PBS (position relative to the SSB site) provided for each pegRNA remain identical for their epegRNA counterpart.In PpAPT, all but pegAPT#2 generate a stop codon at the edit position.For the pegAPT#2, the 4bp-insertion generates a frameshift that creates a downstream stop codon at the amino acid position 24 in the first exon.

FFig. S7 .
Fig. S7.Prime Editing quality using pUbi-PPE MMLV and four APT-pegRNAs.Nature of PE for each pegRNA was assessed by PCR sequencing (Primers in TableS3).n: number of edited plants that were sequenced.

Table S4 : Prime Editing efficiency and quality of pegAPT#3 variants using fused and split PE systems in P. patens. Name pUbi-PE a Split PE a nb 2FA R b Mut. Freq. % c PE % (n) d nb 2FA R b Mut. Freq. % c PE % (n) d pegAPT#3
2-FA R stands for the total number of 2-FA resistant plants obtained during the three transfections.cMutationfrequency (Mut.Freq.) is the % of apt mutants (2-FA R ) among the regenerated plants.dPrimeEditing efficiency (PE) is the % of plants with the expected edits among the apt mutants.n for number of sequenced plants.
a Data from 3 independent transfections.b