Highly efficient CRISPR-SaKKH tools for plant multiplex cytosine base editing

Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.

Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Novel Wx alleles generated by base editing for improvement of rice grain quality

Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

The most popular CRISPR-SpCas9 systemrecognizes canonical NGG protospacer adjacent motifs(PAMs).Previously engineered SpCas9 variants,such as Cas9-NG,favor G-rich PAMs in genome editing.In this manuscript,we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis,C to T base editing,and A to G base editing at A-rich PAMs.This study fills amajor technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants,which greatly expands the targeting scope of CRISPR-Cas9.Finally,our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems,facilitating easy adoption of the systems by others.We anticipate that more tools,such as prime editing,homology-directed repair,CRISPR interference,and CRISPR activation,will be further developed based on our promising iSpyMac-Cas9 platform.