SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

Abstract

Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

Efficient generation of homozygous substitutions in rice in one generation utilizing an rABE8e base editor

“…Very recently, three papers were published describing TadA8e fused with different Cas9 variants, including SpRY, SpG, SpCas9‐NRRH, SpCas9‐NRTH and SpCas9‐NRCH for A‐to‐G base editing (Li et al, 2020; Ren et al, 2021; Xu et al, 2021). In agreement with our results, TadA8e showed high editing efficiencies in these different systems, especially when fused with SpG, SpCas9‐NRRH, SpCas9‐NRTH, and SpCas9‐NRCH, and its homozygous substitution rate was as high as 66.7% for a specific target (Li et al, 2020).…”

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

“…Among engineered Cas9 variants, the near-PAMless nucleases, SpG and especially SpRY variants, present the most relaxed PAM requirements to date. The activity of these nucleases has been well described in human cell lines 6 and more recently in plants [33][34][35][36] . However, SpG and SpRY have never been applied in animals.…”

“…Furthermore, recent publications have demonstrated not only the capacity of SpG and SpRY to produce indels in mammalian cells and plants and but also their adaptation to base editor systems that take advantage of their relaxed PAMs 6,34,35 . Our optimized CRISPR-SpG and SpRY systems in vivo now set the basis for carrying out base editing and other CRISPR applications in animals [40][41][42] with a greatly improved targeting landscape.…”

Base Editing in Plants: Applications, Challenges, and Future Prospects

“…The researchers further optimized SpG to develop SpRY that has the potential of targeting almost all PAM. Xu et al (2020a) have researched the effectiveness of both SpG and SpRY nucleases against separate PAMs and their use in both cytosine and adenine base editing using transgenic rice callus. They reported that even though SpG recognizes NG PAM sequences, its performance is less when compared to SpCas9-NG in rice.…”