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.

Design and self-assembly of PBLG-b-ELP hybrid diblock copolymers based on synthetic and elastin-like polypeptides.

The precision synthesis and self-assembly of amphiphilic copolypeptides containing a recombinant elastin-like polypeptide (ELP) block used as a macroinitiator for the ring opening polymerization (ROP) of γ-benzyl-l-glutamate (γ-BLG NCA) are herein presented. The molecular weight of the resulting PBLG-b-ELP block copolypeptides was precisely controlled without the use of complex initiator or demanding experimental setup. Diblock copolypeptides were obtained with an excellent control of the polymerization highlighted by the dispersity below 1.04. These amphiphilic hybrid synthetic/recombinant copolypeptides were self-assembled in water and the nanoparticles obtained were characterized by a combination of dynamic light scattering and electron microscopy. A variety of morphologies, namely polymersomes, inter-connected worm-like micelles and spherical micelles, were evidenced depending on the hydrophilic ratio of the diblocks as well as the self-assembly procedure.

Physcomitrium patens Protoplasting and Protoplast Transfection.

Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata. Additionally, we detail procedures for their transfection, their plating for optimal regeneration, and three alternative selection approaches. To improve the consistency of protoplast regeneration, we describe a new option for protoplast embedding. The use of an alginate matrix to regenerate moss protoplast alleviates the use of warm agarized medium. Thus, it optimizes transformed protoplast survival without any morphological detrimental effect or impact on transfection efficiency.

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.