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Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available now. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1,500 folds. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e., G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base-editing approach by endowing the engineered DNA glycosylase the capability to selectively excising a new type of substrate.

SUMMARYInterspecies organogenesis via blastocyst complementation provides a unique platform to study development in an evolutionarily context and holds potential to overcome world-wide organ shortages1. By using this technique, rat pancreas, thymus, heart, and eye tissues have been generated in mice2–4. To date, however, xeno-generation of brain tissues has not been achieved through blastocyst complementation. Here, we developed an optimized one-step blastocyst complementation strategy based on C-CRISPR5, which facilitated rapid screening of candidate genes to support blastocyst complementation. Among the seven WNT pathway-related genes selected for targeting, onlyDkk1orHesx1deficiency supported forebrain complementation by blastocyst injection of mouse embryonic stem cells (mESCs). Further, injecting rat ESCs (rESCs) into mouse blastocysts deficient forHesx1but notDkk1supported the development of adult chimeric forebrains comprised a large proportion of rat cells that were structurally and functionally similar to the mouse forebrains. Our analysis revealed that the rESC-derived forebrains developed along the spatial-temporal trajectory with the mouse forebrains rather than rat forebrains, but gene expression profiles of rESC-derived nerve cells surprisingly maintained the characteristics of the rat cells. We noted that the chimeric rate gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late prenatal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRIPSR based IBC strategy developed here holds great potential to broaden the study and application of interspecies organogenesis.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and non-carriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus (SLE) and familial hypercholesterolemia (FH). Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

Competitive coevolution between microbes and viruses has led to the diversification of CRISPR-Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR-Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research.

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences., DNA base editors can display off-target effects on DNA and RNA. Here the authors embed the base editing enzymes in the middle of nCas9 to reduce these without impacting on-target editing.

CRISPR-Cas13 systems have recently been employed for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has imposed a major barrier for their in vivo applications. We designed a dual-fluorescent reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants (including Cas13d and Cas13X) exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. Importantly, high-fidelity Cas13 variants show comparable RNA knockdown activity with wild-type Cas13 but no detectable collateral damage in transgenic mice and adeno-associated virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effect are now available for targeted degradation of RNAs in basic research and therapeutic applications.

The early maize (Zea mays) seed undergoes several developmental stages after double fertilization to become fully differentiated within a short period of time, but the genetic control of this highly dynamic and complex developmental process remains largely unknown. Here, we report a high temporal-resolution investigation of transcriptomes using 31 samples collected at an interval of 4 or 6 h within the first six days of seed development. These time-course transcriptomes were clearly separated into four distinct groups corresponding to the stages of double fertilization, coenocyte formation, cellularization, and differentiation. A total of 22,790 expressed genes including 1415 transcription factors (TFs) were detected in early stages of maize seed development. In particular, 1093 genes including 110 TFs were specifically expressed in the seed and displayed high temporal specificity by expressing only in particular period of early seed development. There were 160, 22, 112, and 569 seed-specific genes predominantly expressed in the first 16 h after pollination, coenocyte formation, cellularization, and differentiation stage, respectively. In addition, network analysis predicted 31,256 interactions among 1317 TFs and 14,540 genes. The high temporal-resolution transcriptome atlas reported here provides an important resource for future functional study to unravel the genetic control of seed development.

Broomcorn millet (Panicum miliaceum L.) has strong tolerance to abiotic stresses, and is probably one of the oldest crops, with its earliest cultivation that dated back to ca. ~10,000 years. We report here its genome assembly through a combination of PacBio sequencing, BioNano, and Hi-C (in vivo) mapping. The 18 super scaffolds cover ~95.6% of the estimated genome (~887.8 Mb). There are 63,671 protein-coding genes annotated in this tetraploid genome. About ~86.2% of the syntenic genes in foxtail millet have two homologous copies in broomcorn millet, indicating rare gene loss after tetraploidization in broomcorn millet. Phylogenetic analysis reveals that broomcorn millet and foxtail millet diverged around ~13.1 Million years ago (Mya), while the lineage specific tetraploidization of broomcorn millet may be happened within ~5.91 million years. The genome is not only beneficial for the genome assisted breeding of broomcorn millet, but also an important resource for other Panicum species., Broomcorn millet is one of the oldest crops cultivated by human that has strong abiotic stress tolerance. To facilitate genome assisted breeding of this and related species, the authors report its genome assembly and conduct comparative genome structure and evolution analyses with foxtail millet.

Recently emerging SARS-CoV-2 virus has caused a global pandemic, with millions of infections and over 200, 000 deaths1. However, development of effective anti-coronavirus treatments has lagged behind. Competitive co-evolution between microbes and viruses has led to the diversification of microbe’s CRISPR/Cas defense systems against infectious viruses2,3. Among class-2 single effector systems, Cas13 is effective in combating RNA phages4. Previous studies have discovered novel Cas9 and Cas12 systems from metagenomic sequence of natural microbes5-7. Here we report the identification of two additional compact Cas13 families from natural microbes that are effective in degrading RNA viruses in mammalian cells. Using metagenomic terabase data sets, we searched for previously uncharacterized Cas13 genes proximal to the CRISPR array with a customized computational pipeline, and identified two most compact families (775 to 803 amino acids) of CRISPR-Cas ribonucleases, named hereafter as CRISPR/Cas type VI-E and VI-F. Out of seven Cas13 proteins, we found that Cas13e.1 was the smallest and could be engineered for efficient RNA interference and base editing in cultured mammalian cell lines. Moreover, Cas13e.1 has a high activity for degrading SARS-CoV-2 sequences and the genome of live influenza A virus (IAV). Together with a minimal pool of 10 crRNAs, Cas13e.1 could target over 99% of all known 3,137 coronavirus genomes for achieving antiviral defense. Overall, our results demonstrated there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, thus potentially useful for preventing viral infection in humans such as COVID-19.

The smallest Cas13 family protein, CasRx, has a high cleavage activity and targeting specificity, offering attractive opportunity for therapeutic applications. Here we report that delivery of CasRx by adeno-associated virus via intravitreal injection could efficiently knockdown Vegfa transcripts and significantly reduce the area of laser-induced choroidal neovascularization in a mouse model of age-related macular degeneration. Thus, RNA-targeting CRISPR system could be used for in vivo gene therapy.

RNA knockdown in vivo carries significant potential for disease modelings and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of the genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro. Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells. However, the RNA-targeting activity of Cas13d still needs to be verified in vivo. In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.

Summary Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Muller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson’s disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.

Starting from nitrate aqueous solutions with citrate acid and polyethylene glycol (PEG) as additives, YVO 4 :Eu phosphors were prepared by a two-step spray pyrolysis (SP) and citrate-gel (CG) methods, respectively. The morphology and luminescence properties of YVO 4 :Eu phosphors prepared from SP method were investigated by changing the concentration of precursor solution, the concentration of PEG and the annealing temperatures, respectively. The emission intensity of the phosphors increased with increasing the annealing temperature and solution concentration due to the increase of the crystallinity and particles size. Certain PEG concentration was necessary for obtaining solid spherical particles, and the optimum emission could be observed when the concentration of PEG was 0.02 g/ml. Compared with the spherical YVO 4 :Eu phosphors prepared by SP method, the CG-derived non-spherical YVO 4 :Eu phosphors showed lower emission intensity when annealed at 1000 and 1200 °C. The optimum concentration for the luminescence of Eu 3+ is determined to be 7 mol % of Y 3+ in YVO 4 host.