The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants. Supplementary Information The online version contains supplementary material available at 10.1007/s11248-024-00381-1.


Introduction
Genome editing is the deliberate and targeted manipulation of genetic materials in order to alter their information.Ever since the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas system was reported, continuous improvements have been made allowing highly targeted editing of the genomes for basic and applied scientific research in many species, including plants (Hassan et al. 2021).However, limitations exist, one of them being due to the necessity for the CRISPR-Cas immune systems to distinguish self from nonself.For this, the Cas nucleases require a specific protospacer-adjacent motif (PAM) close to the target to be active.This requirement for Cas nucleases to recognize a specific PAM restrains the application of this technology for genome editing.For the most widely used SpCas9, this PAM corresponds to NGG, with a low tolerance for NAG and NGA PAMs.Several groups have worked to decrease or even remove the PAM restriction of Cas9 and highly flexible PAM spCas9, such as SpCas9-NG, xCas9, XNG-Cas9, or SpRY, have been used in crop plants like rice and tomato, and in one model plant, Arabidopsis (see Vol:. ( 1234567890) Hassan et al. 2021 for a review).Notably, a SpRY variant was designed to have a highly relaxed PAM requirement (Walton et al. 2020).This near-PAMless variant of the Cas9 allowed for the generation of previously unattainable genetic variations in fungi and animals, but also in the plants Arabidopsis and rice (Li et al. 2021;Pan et al. 2021;Ren et al. 2021;Xu et al. 2021;Wu et al. 2022).
These near-PAMless Cas9 nucleases have increased significantly the number of sites that could be targeted, however, in general, efficiency of these variants does not match the efficiency of the original SpCas9 with a canonical NGG PAM.Various studies aimed at increasing efficiency of the Cas9 nucleases by modifying different structural parameters, such as the number of nuclear localization signals, the codon usage and the presence of introns in the coding sequence of the Cas9 gene (Hassan et al. 2021).In particular, a study showed that the addition of Arabidopsis introns in the sequence of the coding sequence of a Zea mays codon optimized Cas9 improved significantly the editing efficiency in Arabidopsis and other dicotyledons (Grützner et al. 2021).The same study also showed that the presence of two NLS in the construct allowed for a better nuclear import, thus a better mutagenetic activity of the nuclease.
Because efficiency of editing for a given PAM by these near-PAMless Cas9 can vary from one species to another (Hassan et al. 2021), we decided to evaluate the efficiency of editing via the SpRY variant in the model plant Physcomitrium patens.To optimize SpRY Cas9 activity, we incorporated six introns in the SpRY Cas9 coding sequence and added NLS sequences in both the N-terminal and the C-terminal of the nuclease.Our work reveals that SpRYCas9i has varying efficiency with different PAMs, showing higher recognition of certain PAMs and comparable mutation patterns to SpCas9.Notably, this SpRYCas9i variant favored NRN PAMs similar to what was observed in human cells and zebrafish.Finally, we show that the SpRYCas9i's offtarget activity was minimal in Physcomitrium patens.

Plant material and culture
We used the Physcomitrium patens ecotype Gransden pedigree Versailles (Haas et al. 2020) in this study.
Tissue was routinely maintained and propagated on PpNH4 medium either by tissue picking or through tissue blending in sterile purified water (Schaefer et al. 1991).Culture chamber conditions were set at 60% humidity, temperature at 22 °C with a long day light cycle of 16 h of light (quantum irradiance of 80 μmol m −2 s −1 ) and 8 h of dark.

Moss transfection and selection procedures
Moss protoplast isolation was performed from six day-old blended protonemal tissue as previously described (Charlot et al. 2022).180 000 protoplasts were transfected with a total amount of 10 µg of circular plasmid DNA per transfection.After transfection, the dilution of the transfection reaction, its embedding in alginate and spreading on cellophane disks laid atop of PpNH4 medium supplemented with 0.33 M Mannitol were performed as previously described (Charlot et al. 2022).Plants on cellophane disks were then selected on PpNH4 supplemented with 10 μM 2-FA (Fluorochem) to select clones that were mutated at the APT locus (Collonnier et al. 2017).Growing plants after ten days were counted and individually sub-cultured on fresh PpNH4 medium until harvesting for genotyping.
PCR and sequence analysis of the edited plants Moss genomic DNA samples were isolated from 50 mg of fresh tissue in 96-well microtube plates as previously described (Lopez-Obando et al. 2016).Molecular analysis of the mutations induced in the APT gene was based on Sanger sequencing (Genoscreen, Lille, France) of PCR fragments using primers (Table S2) flanking the targeted APT locus.Predicted potential off-target loci (Table S3) were identified using the webtool CRISPOR and their molecular analysis was based on Sanger sequencing of PCR fragments using primers (Table S2) surrounding the identified loci.

Graphics and statistical analysis
Graphs were generated with Excel.The Shapiro-Wilk test has been performed to evaluate replicate normality.The Levene's test was used to evaluate variance homogeneity across the experiment.Subsequently, a paired t-test was performed to compare the sgRNA guide efficiency.All tests were performed in R studio (R 4.2.1)(Table S4, S5 and S6).Samples with mutation rate equal to 0 were excluded from the statistical analysis.

Results and discussion
Efficiency of genome editing in plants using the SpRY variant of the SpCas9 is generally lower compared to the original SpCas9 (Li et al. 2021;Ren et al. 2021).In order to improve the efficiency of SpRY we carried out optimization of its expression and developed a PAM-less Cas9 variant that we named SpRY-Cas9i.The SpRYCas9i gene contains the modifications described in Walton et al 2020, introns based on the zCas9i (Grützner et al. 2021) and two NLS targeting signals, a SV40 and a nucleoplasmin NLS sequences (Fig. 1).
To assess the edition efficiency of SpRYCas9i, we used the APT gene as a reporter of the genome editing.In short, the APT gene codes for the APRT enzyme, a Type I phosphoribosyltransferase family involved in the conversion process of adenine to adenosine monophosphate (AMP) in normal conditions.This APRT enzyme also has the capacity to convert adenine analogues such as the 2-Fluoroadenine (2-FA) into 2-Fluoro-AMP, a lethal compound for plants (Fig. 2a).This property can be used for counter-selection of the non-edited individuals as they will not be able to survive on a 2-FA-containing medium.To test the editing efficiency of SpRYCas9i for different PAMs, we designed 16 gRNAs targeting the APT gene with PAMs containing all possible nucleotide combinations in the second and third position (Fig. 2b).P. patens wild-type protoplasts were co-transfected by PEG-mediated transformation with two plasmids, one bearing the SpRYCas9i gene under the control of the maize Ubi promoter (pUbi_SpRY-Cas9i, Fig. 1, Fig. S1) and another bearing one of the 16 sgRNAs under the control of a P. patens U6 promoter.As a control, we used the native SpCas9 under the control of the maize Ubi promoter (pUbi_SpCas9, Fig. S2) with the same 16 sgRNAs.The relative mutation rates (expressed in percentages), using the different sgRNAs, were estimated by dividing the number of 2-FA-resistant plants by the number of regenerating plants observed just before the transfer on 2-FA.
Mutation rate using the native SpCas9 and a sgRNA targeting a NGG PAM is close to 13% (Table 1).As expected, mutation rate of sgRNAs using a non NGG PAM is very low or null, with only one sgRNA, using a nAG PAM, leading to an important number of apt  mutants (Table 1).Like the SpCas9, SpRYCas9i is recognizing the nGG PAM, but less efficiently (sixfold less) than the native Cas9 (Fig. 3, Table 2).The PAMs for which the efficiency of SpRYCas9i is the highest are NAA and NAC, which are not recognized at all by the SpCas9 (Table 1).In general, NRN PAMs are recognized significantly more efficiently by the SpRYCas9i compared to NYN PAMs (Fig. 3 and Table S5 for p-value of paired t-test, p-value ≤ 0.05 considered as significant).Interestingly, the sgRNA target APT locus containing the PAM that corresponds to the three first base pairs of the RNA scaffold, GTT, is not or very weakly mutated by the SpRYCas9i in P. patens.This is probably due to the detrimental self-target effect on the sgRNA sequence that could be observed with SpRY and SpCas9-NG, in rice (Qin et al. 2020;Xu et al. 2021).
In order to characterize the mutations generated by the SpRYCas9i, we amplified by PCR and sequenced the APT gene in 62 independent mutant plants obtained with the different sgRNAs (Fig. S3).As expected, all the mutations were located in the vicinity of the PAM targets of the SpRYCas9iinduced cleavage site and generated loss of APT function.These mutations consisted mainly of deletions and a few insertions, both with or without substitutions.Interestingly, for a majority of the deletions (53%), microhomologies (of 2-4 bp) could be detected between the end of the deletion itself and the sequence located just upstream of the deletion  (Fig. S3).These different patterns of mutations are very similar to the ones already observed with the SpCas9 in P. patens (Collonnier et al. 2017), with a strong part of the deletions that could be explained by alt-EJ-mediated repair based on microhomologies (Oh and Myung 2022).
The relaxed PAM tolerances of near-PAMless Cas9 variants can, in principle, lead to recognition of additional off-target sites in the genome and SpRY has been shown to exhibit increased off-targeting compared to the native SpCas9 that could be decreased by using an HF1 version of SpRY (Walton et al. 2020).In order to evaluate the off-target activity of the SpRY-Cas9i in P. patens, off-target candidate loci (Table S3) were amplified and sequenced in 10 apt mutant plants generated with one sgRNA targeting a NRN type PAM (13 potential off-targets) and 10 apt mutant plants generated with one sgRNA targeting a NYN type PAM (9 potential off-targets).No mutation could be detected in the potential off-target sequences for any of the 20 tested plants.
We can conclude from these data that the structurally engineered SpRY is capable of recognizing NRN and several NYN PAMs in P. patens, but with a reduced efficiency with the latter.Mutations produced by SpRYCas9i are of the same nature as the ones produced by the SpCas9.The preference of SpRY-Cas9i for NRN PAMs mirrors what has been already observed in human cells, zebrafish, and rice (Walton et al. 2020;Xu et al. 2021;Liang et al. 2022).The near-PAMless variants have already been adapted into base editors and prime editor and should be adaptable to base editors and prime editors used in P. patens (Perroud et al. 2021;Guyon-Debast et al. 2021).

Fig. 2
Fig. 2 Schematic description of the APT reporter gene and positions of the sgRNAs.a The APT gene encodes the Adenine phosphoribosyltransferase (APRT) catalyses a phosphoribosyl transfer from Phosphoribosyl Pyrophosphate (PRPP) to adenine, forming adenosine monophosphate (AMP) and releasing pyrophosphate (PPi).In the presence of 2-Fluoroadenine APRT will form 2-FluoroAMP, a toxic compound for the cell.

Fig. 3
Fig. 3 Editing of the APT reporter gene at NYN and NRN PAM targets.Mutation efficiency of the APT (Adenine phosphoribosyltransferase) gene using guides targeting NYN and NRN PAM (protospacer adjacent motif) targets, with SpRY-Cas9i.Relative mutation efficiency expresses the frequency

Table 1
Mutation rates of the APT gene for different PAM using the native Cas9 Vol.: (0123456789)