Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Nuclear translocation of OsMFT1 that is impeded by OsFTIP1 promotes drought toleranee in rice

Drought is the leading environmental threat affecting crop productivity,and plants have evolved a series of mechanisms to adapt to drought stress.The FT-interacting proteins(FTIPs)and phosphatidylethanola mine.binding proteins(PEBPs)play key roles in developmental processes,whereas their roles in the regulation of stress response are still largely unknown.Here,we report that OsFTIP1 negatively regulates drought response in rice.We showed that OsFTIP1 interacts with rice MOTHER OF FT AND TFL1(OsMFT1),a PEBP that promotes rice tolerance to drought treatment.Further studies discovered that OsMFT1 interacts with two key drought-related transcription factors,OsbZIP66 and OsMYB26,regulating their binding capacity on drought-related genes and thereby enhancing drought toleranee in rice.Interestingly,we found that OsFTIP1 impedes the nucleocytoplasmic translocation of OsMFT1,implying that dynamic modulation of drought-responsive genes by the OsMFT1-OsMYB26 and OsMFT1-OsbZIP66 complexes is integral to OsFTIP1-modulated nuclear accumulation of OsMFT1.Our findings also suggest that OsMFT1 might act as a hitherto unknown nucleocytoplasmic trafficking signal that regulates drought tolerance in rice in response to environmental signals.

Plant Genome Editing Using FnCpfl and LbCpf Nucleases at Redefined and Altered PAM-Sites

Dear Editor CRISPR from Prevotella and Francisella 1 （Cpfl） is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif （PAM） for DNA targeting （Zetsche et al., 2015）. CRISPR-Cpfl has unique features that could be advantageous over the CRISPR-Cas9 system. For example, Cpfl requires only a 42 nt crRNA, while Cas9 uses 100 nt gRNA. While Cas9 generates blunt ends of DNA breaks, the Cpfl cleavage results in 5＇ overhangs distal from the protospacer, which may improve efficiency for NHEJ-based gene insertion. Interestingly, Cpfl proteins also have RNase activity （Fonfara et al., 2016）, which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants （Wang et al., 2017; Zetsche et al., 2017）.

The OsFTIP6-OsHB22-OsMYBR57 module regulates drought response in rice

Plants have evolved a sophisticated set of mechanisms to adapt to drought stress.Transcription factors play crucial roles in plant responses to various environmental stimuli by modulating the expression of numerous stress-responsive genes.However,how the crosstalk between different transcription factor families orchestrates initiation of the key transcriptional network and the role of posttranscriptional modification of transcription factors,especially in cellular localization/trafficking in response to stress in rice,remain still largely unknown.In this study,we isolated an Osmybr57 mutant that displays a drought-sensitive phenotype through a genetic screen for drought stress sensitivity.We found that OsMYBR57,an MYB-related protein,directly regulates the expression of several key drought-related OsbZ/Ps in response to drought treatment.Further studies revealed that OsMYBR57 interacts with a homeodomain transcription factor,OsHB22,which also plays a positive role in drought signaling.We further demonstrate that OsFTIP6 interacts with OsHB22 and promotes the nucleocytoplasmic translocation of OsHB22 into the nucleus,where OsHB22 cooperates with OsMYBR57 to regulate the expression of drought-responsive genes.Our findings have revealed a mechanistic framework underlying the OsFTIP6-0sHB22-0sMYBR57 module-mediated regulation of drought response in rice.The OsFTIP6-mediated OsHB22 nucleocytoplasmic shuttling and OsMYBR57-0sHB22 regulation of OsbZIP transcription ensure precise control of expression of OsLEA3 and Rab21,and thereby regulate the response to water deficiency in rice.