Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Abstract

As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools

“…These results are inconsistent with those obtained in mammalian cells, in which CGBE systems significantly reduced C-to-T substitution and improved C-to-G transversion without excessive indel byproducts [ 20 ]. However, our results are almost consistent with those of studies on rice and poplar [ 22 , 23 ]. The allelic variations in CGBE-rUNG and CGBE-hUNG are mainly biallelic mutations and heterozygous mutations, with homozygous mutations in a small part ( Figure 1 d, Figures S1 and S2 ).…”

“…Based on the principle of BER, the uracil DNA glycosylase inhibitor (UGI) domain in the CBE system is replaced by the UNG domain, and high-efficiency C-to-G transversion is produced in mammalian cells [ 20 , 21 ]. Although studies have evaluated CGBE editing in protoplasts and stable transformations in rice and poplar [ 22 , 23 ], the target sites and data are insufficient to objectively assess the editing performance of CGBE in plants.…”

Efficient C‐to‐G editing in rice using an optimized base editor

“…Genetic analysis of 11 lines confirmed a heritability ranging from 4.2% to 62.5% (Table S2). Our CGBE showed greater stability and higher editing efficiency in rice than the recently reported systems (Sretenovic et al ., 2021). Compared to that in mammalian cells, it gave higher indel frequencies (111 lines, 29.5%, Figure S2), indicating that our CGBE needs to be further optimized in the future by inhibiting the base excision repair process.…”

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

“…Overall, these new precise nucleotide-editing strategies could further expand the CRISPR-based applications for the development of novel quantitative traits with a gain-of-function mutation [184,185]. In addition to its numerous advantages over conventional CRISPR-based systems, there are still certain aspects (e.g., on-target editing efficiency, unwanted mutations, optimal experiment design [186]) that need to be improved further for a more efficient and robust genome editing application.…”