LAUSR

A large number of beneficial agronomic traits in crops are associated with single nucleotide polymorphisms (SNPs) or point mutations (Jiao et al., 2010; Li et al., 2017; Ma et al., 2015). In the past, site-specific point mutations in a target gene can only be achieved through the CRISPR/Cas9mediated gene replacement via the homology-directed repair (HDR). However, the intrinsically low HDR activity in plant cells and the lack of efficient way to supply abundant HDR templates in plant nucleus have greatly limited the success rate of gene replacement in plants (Ran et al., 2017). In addition, DNA double-strand breaks (DSBs) generated by the Cas9 nuclease prior to HDR may lead to complicated and unexpected genome disturbance (Kosicki et al., 2018). Since 2016, cytosine base editors (CBEs) have emerged as a new class of tools for introducing precise C-to-T or G-to-A substitutions in eukaryotic genomes without the requirement of HDR template and DSB generation (Chen et al., 2017; Ren et al., 2017). CBEs are hybrid proteins consisting of a cytidine deaminase, a Cas9 nikase (nCas9), and a uracil glycosylase inhibitor (UGI)(Komor et al., 2016; Nishida et al., 2016). To date, different CBEs mainly differ on the cytidine deaminase moiety. Well-characterized cytidine deaminases suitable for building CBEs include the rat APOBEC1 (Komor et al., 2016), sea lamprey CDA1 (Nishida et al., 2016), and human AID (Ma et al., 2016). However, current CBEs induce targeted C-to-T mutagenesis only in a narrow window (i.e., around 5-nt) within the target sequence, and the most frequently used CBE, namely the APOBEC1-nCas9-UGI (also known as BE3 (Komor et al., 2016) or PBE (Zong et al., 2017)), is inefficient for editing cytosines in the context of GC dinucleotide (Komor et al., 2016). Very recently, a study by Zong and colleagues published in Nature Biotechnology has demonstrated efficient base editing in wheat, rice, and potato plants through a novel CBE known as A3A-PBE, which has a much broader editing window and has no bias on the target sequence context (Zong et al., 2018). In the report by Zong et al., the authors utilized the human deaminase APOBEC3A to replace the rat APOBEC1 in PBE (Zong et al., 2017) to create the new base editor A3A-PBE. To quickly evaluate the C-to-T mutagenesis induced by A3A-PBE, they adopted a fluorescent reporter system, in which successful C-to-T conversions could be mirrored by the conversion of BFP to GFP. These assays indicated that A3A-PBE could induce desired base editing at a much higher efficiency than PBE. The authors further targeted 20 endogenous genomic loci by A3A-PBE in wheat, rice, and potato protoplasts and examined the editing outcomes through deep sequencing of the genomic PCR products of the target regions. These assays again revealed that on average A3A-PBE exhibits a C-to-T editing efficiency more than 10fold higher than PBE. The deep sequencing results also suggested that A3A-PBE enables a 17-nt editing window from the protospacer position 1 to 17, which is much wider than that of PBE (Zong et al., 2017). Moreover, A3A-PBE was found to induce base substitutions with high product purity and low frequency of indels. The A3A-PBE was also