Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion

Abstract

Nucleotide base editors in plants have been limited to conversion of cytosine to thymine. Here, we describe a new plant adenine base editor based on an evolved tRNA adenosine deaminase fused to the nickase CRISPR/Cas9, enabling A•T to G•C conversion at frequencies up to 7.5% in protoplasts and 59.1% in regenerated rice and wheat plants. An endogenous gene is also successfully modified through introducing a gain-of-function point mutation to directly produce an herbicide-tolerant rice plant. With this new adenine base editing system, it is now possible to precisely edit all base pairs, thus expanding the toolset for precise editing in plants.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1443-z) contains supplementary material, which is available to authorized users.

An adenine base editor with expanded targeting scope using SpCas9‐NGv1 in rice

“…Two types of base editors have now been developed: cytosine base editors (CBEs)-cytidine deaminase fused with catalytically impaired Cas9-can efficiently convert a C-G base pair to a T-A pair, while adenine base editors (ABEs)-Cas9 nickase (nCas9) plus engineered Escherichia coli adenosine deaminase (TadA)enable A-T to G-C conversions (Gaudelli et al, 2017;Kim, 2018). Both CBEs and ABEs have been applied successfully in plants such as Arabidopsis, wheat and rice (e.g., Hua et al, 2018Hua et al, , 2019Li et al, 2018). However, potential base editing targets remain restricted by the requirement for protospacer adjacent motif (PAM) sequences around these sites.…”

“…To extend Cas9-recognized sites, several groups have engineered Cas9 variants with amino acid substitutions and modified PAM sequences. Using these Cas9 variants in base editors could expand the scope of base editing sites (Endo et al, 2019;Hu et al, 2018;Hua et al, 2019;Nishimasu et al, 2018). Engineered SpCas9s, SpCas9-NG variants and xCas9 variants that can recognize NG as PAM have been reported recently (Hu et al, 2018;Nishimasu et al, 2018).…”

“…Using these Cas9 variants in base editors could expand the scope of base editing sites (Endo et al, 2019;Hu et al, 2018;Hua et al, 2019;Nishimasu et al, 2018). Engineered SpCas9s, SpCas9-NG variants and xCas9 variants that can recognize NG as PAM have been reported recently (Hu et al, 2018;Nishimasu et al, 2018). SpCas9-NG and xCas9 variants can induce mutation at target sites with NG PAMs in rice (Endo et al, 2019;Wang et al, 2019), while the in vitro DNA cleavage activity of SpCas9-NGv1 (reported as ARVRFRR; Nishimasu et al, 2018), which includes a 7-aa mutation (R1335A/L1111R/D1135V/ G1218R/E1219F/A1322R/T1337R) in the PAM-interacting domain, is higher than that of xCas9 (Endo et al, 2019;Nishimasu et al, 2018).…”

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

“…With the sgRNA providing targetspecificity, the dCas9-base editors can create point mutations in the target DNA with high specificity and precision. This new technology was quickly adopted in plants, including watermelon (Lu and Zhu 2017;Li et al 2018a;Ren et al 2018;Tian et al 2018). Table 1 summarizes published reports of genome editing in fruit crops.…”

CRISPR/Cas9‐Based Genome Editing and its Applications for Functional Genomic Analyses in Plants

“…Through seven rounds of extensive directed evolution and protein engineering of the bacterial tRNA adenine deaminase TadA, researchers produced an adenine base editor (ABE7.10), which efficiently converts AT to GC base pairs in a programmable manner with very high product purity in human cells . Several independent groups have reported that the ABE7.10 editor efficiently induced AT to GC base pairs at a broad range of endogenous target sites in the rice genome …”