BackgroundTargeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date.ResultsWe conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation.ConclusionsOur comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1458-5) contains supplementary material, which is available to authorized users.

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Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice ( L. ) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr-facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome and might mark other cis-regulatory DNA elements that are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants.

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at Arich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/ Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

Layer-by-layer polyelectrolyte adsorption in porous polymeric membranes provides a simple way to create ion-exchange sites without greatly decreasing hydraulic permeability (<20% reduction in permeability). At 80% breakthrough, membranes coated with 3-bilayer poly(styrene sulfonate) (PSS)/polyethyleneimine (PEI) films bind 37±6 mg of negatively charged Au colloids per mL of membrane volume. The binding capacity of membranes coated with 1-bilayer films decreases in the order PSS/PEI>PSS/poly(diallyldimethyl ammonium chloride)>PSS/poly(allylamine hydrochloride). Films terminated with a polyanion present cation-exchange sites that bind lysozyme, and the lysozyme-binding capacities of (PSS/PEI)3/PSS films increase with the ionic strength of the solution from which the last PSS layer is deposited. Charge screening during deposition of the terminal PSS layer gives rise to a larger number of ion-exchange sites and lysozyme binding capacities as high as 16 mg per mL of membrane. At 10% breakthrough, a stack of 3 membranes binds 3 times as much lysozyme as a single membrane, showing that stacking is an effective way to increase capacity.

This article discusses the relationship between the molecular structure of bolaamphiphiles bearing mesogenic groups and their interfacial self-organized morphology. On the basis of the molecular structures of bolaamphiphiles, we designed and synthesized a series of molecules with different hydrophobic alkyl chain lengths, hydrophilic headgroups, mesogenic groups, and connectors between the alkyl chains and the mesogenic group. Through investigating their interfacial self-organization behavior, some experiential rules are summarized: (1) An appropriate alkyl chain length is necessary to form stable surface micelles; (2) different categories of headgroups have a great effect on the interfacial self-organized morphology; (3) different types of mesogenic groups have little effect on the structure of the interfacial assembly when it is changed from biphenyl to azobenzene or stilbene; (4) the orientation of the ester linker between the mesogenic group and alkyl chain can greatly influence the interfacial self-organization behavior. It is anticipated that this line of research may be helpful for the molecular engineering of bolaamphiphiles to form tailor-made morphologies.