Targeted, programmable, and precise tandem duplication in the mammalian genome.

Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome. In this strategy, we design a pair of in trans prime editing guide RNAs (pegRNAs) for each targeted tandem duplication, which encode the same edits but prime the single-stranded DNA (ssDNA) extension in opposite directions. The reverse transcriptase (RT) template of each extension is designed homologous to the target region of the other single guide RNA (sgRNA) to promote the reannealing of the edited DNA strands and the duplication of the fragment in between. We showed that TD-PE produced robust and precise in situ tandem duplications of genomic fragments ranging from ∼50 bp to ∼10 kb, with a maximal efficiency up to 28.33%. By fine-tuning the pegRNAs, we achieved simultaneous targeted duplication and fragment insertion. Finally, we successfully produced multiple disease-relevant tandem duplications, showing the general utility of TD-PE in genetic research.

Significantly Accelerated Photosensitized Formation of Atmospheric Sulfate at the Air-Water Interface of Microdroplets.

The multiphase oxidation of sulfur dioxide (SO2) to form sulfate is a complex and important process in the atmosphere. While the conventional photosensitized reaction mainly explored in the bulk medium is reported to be one of the drivers to trigger atmospheric sulfate production, how this scheme functionalizes at the air-water interface (AWI) of aerosol remains an open question. Herein, employing an advanced size-controllable microdroplet-printing device, surface-enhanced Raman scattering (SERS) analysis, nanosecond transient adsorption spectrometer, and molecular level theoretical calculations, we revealed the previously overlooked interfacial role in photosensitized oxidation of SO2 in humic-like substance (HULIS) aerosol, where a 3-4 orders of magnitude increase in sulfate formation rate was speculated in cloud and aerosol relevant-sized particles relative to the conventional bulk-phase medium. The rapid formation of a battery of reactive oxygen species (ROS) comes from the accelerated electron transfer process at the AWI, where the excited triplet state of HULIS (3HULIS*) of the incomplete solvent cage can readily capture electrons from HSO3- in a way that is more efficient than that in the bulk medium fully blocked by water molecules. This phenomenon could be explained by the significantly reduced desolvation energy barrier required for reagents residing in the AWI region with an open solvent shell.

Rapid Sample Pretreatment Facilitating SERS Detection of Trace Weak Organic Acids/Bases in Simple Matrices.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for highly sensitive qualitative and quantitative analyses of trace targets. However, sensitive SERS detection can only be facilitated with a suitable sample pretreatment in fields related to trace amounts for food safety and clinical diagnosis. Currently, the sample pretreatment for SERS detection is normally borrowed and improved from the ones in the lab, which yields a high recovery but is tedious and time-consuming. Rapid detection of trace targets in a complex environment is still a considerable issue for SERS detection. Herein, we proposed a liquid-liquid extraction method coupled with a back-extraction method for sample pretreatment based on the pH-sensitive reversible phase transition of the weak organic acids and bases, where the lowest detectable concentrations were identical before and after the pretreatment process. The sensitive (µg L-1 level) and rapid (within 5 min) SERS detection of either koumine, a weak base, or celastrol, a weak acid, was demonstrated in different drinking water samples and beverages. Furthermore, target generality was demonstrated for a variety of weak acids and bases (2 < pKa < 12), and the hydrophilicity/hydrophobicity of the target determines the pretreatment efficiency. Therefore, the LLE-BE coupled SERS was developed as an easy, rapid, and low-cost tool for the trace detection of the two types of targets in simple matrices, which paved the way toward trace targets in complex matrices.

Nanoplastics Detected in Commercial Sea Salt.

People of all ages consume salt every day, but is it really just salt? Plastic nanoparticles [nanoplastics (NPs)] pose an increasing environmental threat and have begun to contaminate everyday salt in consumer goods. Herein, we developed a combined surface enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) approach that can realize the filtration, enrichment, and detection of NPs in commercial salt. The Au-loaded (50 nm) anodic alumina oxide substrate was used as the SERS substrate to explore the potential types of NP contaminants in salts. SRS was used to conduct imaging and quantify the presence of the NPs. SRS detection was successfully established through standard plastics, and NPs were identified through the match of the hydrocarbon group of the nanoparticles. Simultaneously, the NPs were quantified based on the high spatial resolution and rapid imaging of the SRS imaging platform. NPs in sea salts produced in Asia, Australasia, Europe, and the Atlantic were studied. We estimate that, depending on the location, an average person could be ingesting as many as 6 million NPs per year through the consumption of sea salt alone. The potential health hazards associated with NP ingestion should not be underestimated.

Quantification and Characterization of Fine Plastic Particles as Considerable Components in Atmospheric Fine Particles.

The negative effects of air pollution, especially fine particulate matter (PM2.5, particles with an aerodynamic diameter of ≤2.5 µm), on human health, climate, and ecosystems are causing significant concern. Nevertheless, little is known about the contributions of emerging pollutants such as plastic particles to PM2.5 due to the lack of continuous measurements and characterization methods for atmospheric plastic particles. Here, we investigated the levels of fine plastic particles (FPPs) in PM2.5 collected in urban Shanghai at a 2 h resolution by using a novel versatile aerosol concentration enrichment system that concentrates ambient aerosols up to 10-fold. The FPPs were analyzed offline using the combination of spectroscopic and microscopic techniques that distinguished FPPs from other carbon-containing particles. The average FPP concentrations of 5.6 µg/m3 were observed, and the ratio of FPPs to PM2.5 was 13.2% in this study. The FPP sources were closely related to anthropogenic activities, which pose a potential threat to ecosystems and human health. Given the dramatic increase in plastic production over the past 70 years, this study calls for better quantification and control of FPP pollution in the atmosphere.

Strategies and Challenges of Identifying Nanoplastics in Environment by Surface-Enhanced Raman Spectroscopy.

Nanoplastics (<1000 nm) have been evidenced to be universal in a variety of environmental media. They pose a potential cytotoxicity and health risk due to their tiny size, which allows them to easily penetrate biological barriers and enter cells. Here, we briefly review the various prevalent analytical techniques or tools for identifying nanoplastics, and further move to focus on their advantages and disadvantages. Surface-enhanced Raman spectroscopy (SERS) has been implemented for the identification of individual nanoparticles because of its high sensitivity to molecules and ease of rapid characterization. Therefore, we introduce the SERS technique in the following aspects, (1) principles of SERS; (2) strategies and advances in SERS detection of nanoplastics; and (3) applying SERS to real environmental samples. We put our effort into the summarization of efficient SERS substrates that essentially enable the better detection of nanoplastics, and extend to discuss how the reported nanoplastics pretreatment methodologies can bring SERS analysis to practical applications. A further step moving forward is to investigate the problems and challenges of currently applied SERS detection methods and to look at future research needs in nanoplastics detection employing SERS analysis.

Automatic Identification of Individual Nanoplastics by Raman Spectroscopy Based on Machine Learning.

The increasing prevalence of nanoplastics in the environment underscores the need for effective detection and monitoring techniques. Current methods mainly focus on microplastics, while accurate identification of nanoplastics is challenging due to their small size and complex composition. In this work, we combined highly reflective substrates and machine learning to accurately identify nanoplastics using Raman spectroscopy. Our approach established Raman spectroscopy data sets of nanoplastics, incorporated peak extraction and retention data processing, and constructed a random forest model that achieved an average accuracy of 98.8% in identifying nanoplastics. We validated our method with tap water spiked samples, achieving over 97% identification accuracy, and demonstrated the applicability of our algorithm to real-world environmental samples through experiments on rainwater, detecting nanoscale polystyrene (PS) and polyvinyl chloride (PVC). Despite the challenges of processing low-quality nanoplastic Raman spectra and complex environmental samples, our study demonstrated the potential of using random forests to identify and distinguish nanoplastics from other environmental particles. Our results suggest that the combination of Raman spectroscopy and machine learning holds promise for developing effective nanoplastic particle detection and monitoring strategies.

Significant Acceleration of Photocatalytic CO2 Reduction at the Gas-Liquid Interface of Microdroplets.

Solar-driven CO2 reduction reaction (CO2 RR) is largely constrained by the sluggish mass transfer and fast combination of photogenerated charge carriers. Herein, we find that the photocatalytic CO2 RR efficiency at the abundant gas-liquid interface provided by microdroplets is two orders of magnitude higher than that of the corresponding bulk phase reaction. Even in the absence of sacrificial agents, the production rates of HCOOH over WO3 ⋅ 0.33H2 O mediated by microdroplets reaches 2536 µmol h-1 g-1 (vs. 13 µmol h-1 g-1 in bulk phase), which is significantly superior to the previously reported photocatalytic CO2 RR in bulk phase reaction condition. Beyond the efficient delivery of CO2 to photocatalyst surfaces within microdroplets, we reveal that the strong electric field at the gas-liquid interface of microdroplets essentially promotes the separation of photogenerated electron-hole pairs. This study provides a deep understanding of ultrafast reaction kinetics promoted by the gas-liquid interface of microdroplets and a novel way of addressing the low efficiency of photocatalytic CO2 reduction to fuel.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

miR2119, a Novel Transcriptional Regulator, Plays a Positive Role in Woody Plant Drought Tolerance by Mediating the Degradation of the CkBI-1 Gene Associated with Apoptosis.

Caragana korshinskii, an important vegetation restoration species with economic and ecological benefits in the arid region of northwest China, is characterized by significant drought tolerance. However, the underlying molecular mechanisms by which miRNAs confer this trait in C. korshinskii are unclear. Here, we investigate the effect of CkmiR2119 on drought tolerance and identified its target gene, CkBI-1. A negative correlation of CkmiR2119 and CkBI-1 in both stems and leaves in a drought gradient treatment followed by target gene validation suggest that CkmiR2119 might negatively regulate CkBI-1. Consistently, a decrease in the expression of the CkBI-1 gene was observed after both transient transformation and stable transformation of CkamiR2119 in tobacco (Nicotiana tabacum). Moreover, the physiological analysis of CkamiR2119 and CkBI-1 transgenic plants further indicate that CkmiR2119 can enhance the drought tolerance of C. korshinskii in two aspects: (i) downregulating CkBI-1 expression to accelerate vessel maturation in stems; (ii) contributing to a higher level of CkBI-1 in mesophyll cells to inhibit programmed cell death (PCD). This work reveals that CkmiR2119 can increase plants' drought tolerance by downregulating the expression of CkBI-1, providing a theoretical basis to improve plants' ability to withstand stress tolerance by manipulating miRNAs.

The effect of cold exposure on serum cholesterol is dependent upon ApoE.

BACKGROUND: Several lines of evidence indicate that cold stimulation may not only activate brown adipose tissue (BAT) and the white adipose tissue (WAT), but also regulate the lipid metabolism and influence the development of atherosclerosis. However, the study of cold exposure affecting cholesterol metabolism have opposite results in different experiments, and Apolipoprotein E (ApoE) may play an important role. There is still a lack of complete research to illustrate this problem. METHODS: In this study, we first analyzed and discussed the activation of interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) under cold exposure (4 °C) in male wild-type C57BL/6 J (WT) and ApoE-deficient mice (ApoE-/-) fed high-fat diet (HFD) for 4 weeks. Subsequently, we investigated the effect of cold exposure on blood lipid profiles in both models. We further explored whether cold exposure can reduce serum cholesterol. RESULTS: In both WT and ApoE-/- mice, cold exposure activates iBAT and iWAT, as well as hardly affects eWAT. In WT mice,4 weeks cold exposure (4 °C) reduces serum triglyceride by 28%, cholesterol by 30% and LDL-cholesterol by 63%. In ApoE-/- mice, cold stimulation decreases serum triglyceride by 59%, but increases cholesterol by 20% and LDL-cholesterol by 25%. CONCLUSIONS: Based on these findings, we conclude that cold exposure decreases serum cholesterol is dependent upon the existence of ApoE.

Proteomic Analysis Reveals the Renoprotective Effect of Tribulus terrestris against Obesity-Related Glomerulopathy in Rats.

Tribulus terrestris L. (Zygophyllaceae) (TT) is usually used as a cardiotonic, diuretic, and aphrodisiac, as well as for herbal post-stroke rehabilitation in traditional Chinese medicine. However, little is known about the renoprotective effects of TT on obesity-related glomerulopathy (ORG). In this study, 340 monomeric compounds were identified from TT extracts obtained with ethyl acetate combined with 50% methanol. In vitro, IC50 of TT was 912.01 mg/L, and the appropriate concentration of TT against oxidized-low density lipoprotein (ox-LDL) induced human renal glomerular endothelial cells (HRGECs) was 4 mg/L. TT significantly increased the viability (63.2%) and migration (2.33-fold increase) of HRGECs. ORG model rats were induced by a chronic high-fat diet (45%) for 20 weeks and were then treated with TT extract (2.8 g/kg/d) for 8 weeks. Subsequently, the kidneys were removed and their differentially expressed protein profile was identified using two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF MS. Molecular categorization and functional analysis of bioinformatic annotation suggested that excessive energy metabolism, decreased response to stress and low immunity were the potential etiologies of ORG. After TT administration for 8 weeks, body weight, blood pressure, serum cystatin C and cholesterol were decreased. Additionally, TT significantly enhanced the resistance of rats to ORG, decreased energy consumption and the hemorrhagic tendency, and improved the response to acute phase reactants and immunity. In conclusion, TT may play a protective role against ORG in rats.

[Integrated DNA barcoding database for identifying Chinese animal medicine].

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

Glucose-Activated Switch Regulating Insulin Analog Secretion Enables Long-term Precise Glucose Control in Mice With Type 1 Diabetes.

Genetic modification of non-ß-cells to produce insulin is a promising therapeutic strategy for type 1 diabetes; however, it is associated with issues, including biosafety and precise regulation of insulin supply. In this study, a glucose-activated single-strand insulin analog (SIA) switch (GAIS) was constructed to achieve repeatable pulse activation of SIA secretion in response to hyperglycemia. In the GAIS system, the conditional aggregation domain-furin cleavage sequence-SIA fusion protein was encoded by the intramuscularly delivered plasmid and temporarily kept in the endoplasmic reticulum (ER) because it binds to the GRP78 protein; then, upon hyperglycemia, the SIA was released and secreted into the blood. In vitro and in vivo experiments systematically demonstrated the effects of the GAIS system, including glucose-activated and repeatable SIA secretion, long-term precise blood glucose control, recovered HbA1c levels, improved glucose tolerance, and ameliorated oxidative stress. Additionally, this system offers sufficient biosafety, as evidenced by the assays of immunological and inflammatory safety, ER stress, and histological evaluation. Compared with the viral delivery/expression system, the ex vivo implantation of engineered cells, and the exogenous inducer system, the GAIS system combines the advantages of biosafety, effectiveness, persistence, precision, and convenience, providing therapeutic potential for the treatment of type 1 diabetes. ARTICLE HIGHLIGHTS: We undertook this study to establish a glucose-responsive single-strand insulin analog (SIA) self-supply system in vivo. We sought to determine whether the endoplasmic reticulum (ER) can serve as a safe and temporary repository to store designed fusion proteins and release SIAs under hyperglycemic conditions for efficient blood glucose regulation. The intramuscularly expressed plasmid-encoded conditional aggregation domain-furin cleavage sequence-SIA fusion protein can be temporarily stored in the ER, and the SIA can be released under the stimulation of hyperglycemia, resulting in efficient and long-term regulation of stable blood glucose in mice with type 1 diabetes (T1D). The glucose-activated SIA switch system provides applicable potential for T1D therapy, integrating regulation and monitoring of blood glucose levels.

Prolonged control of insulin-dependent diabetes via intramuscular expression of plasmid-encoded single-strand insulin analogue.

Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative method for continuous insulin supply via intramuscular injection of a designed plasmid encoding the single-strand insulin analogue (SIA), which provides safe, effective and prolonged control of insulin-dependent diabetes. To generate a SIA, a short flexible peptide was alternatively introduced into the natural proinsulin to replace its original long and rigid C-peptide. Then, the synthetic promoter SP301 was used to drive potent and specific expression of SIA in skeletal muscle cells. By combining the Pluronic L64 and low-voltage electropulse (L/E), the specialized gene delivery technique was applied to efficiently transfer the constructed plasmid into skeletal muscle cells via intramuscular injection. Through these efforts, a plasmid-based intramuscular gene expression system was established and improved, making it applicable for gene therapy. The plasmid-expressed SIA showed biological functions that were similar to that of natural insulin. A single L/E-pSP301-SIA administration provided sustained SIA expression in vivo for about 1.5 months. In addition, the diabetic mice treated with L/E-pSP301-SIA were much healthier than those with other treatments. This plasmid-based system was safe for the treatment of diabetes and did not cause immune responses or pathological damage. The results confirmed that, in a mouse model, long-term positive effects were achieved by a single intramuscular L/E-pSP301-SIA injection, which consequently provided reliable experimental basis for its clinical application for the treatment of diabetes mellitus with promising prospects.

Prebiotic synthesis of mineral-bearing microdroplet from inorganic carbon photoreduction at air-water interface.

The origin of life on Earth is an enigmatic and intricate conundrum that has yet to be comprehensively resolved despite recent significant developments within the discipline of archaeology and geology. Chemically, metal-sulfide minerals are speculated to serve as an important medium for giving birth in early life, while yet so far direct evidence to support the hypothesis for the highly efficient conversion of inorganic carbon into praxiological biomolecules remains scarce. In this work, we provide an initial indication that sphalerite, employed as a typical mineral, shows its enormous capability for promoting the conversion of inorganic carbon into elementary biomolecule formic acid (HCOOH) in airborne mineral-bearing aerosol microdroplet, which is over two orders of magnitude higher than that of the corresponding conventional bulk-like aqueous phase medium in the environment (e.g. river, lake, sea, etc.). This significant enhancement was further validated by a wide range of minerals and clays, including CuS, NiS, CoS, CdS, MnS, elemental sulfur, Arizona Test Dust, loess, nontronite, and montmorillonite. We reveal that the abundant interface of unique physical-chemical features instinct for aerosol or cloud microdroplets reduces the reaction energy barrier for the reaction, thus leading to extremely high HCOOH production (2.52 × 1014 kg year-1). This study unfolds unrecognized remarkable contributions of the considered scheme in the accumulation of prebiotic biomolecules in the ancient period of the Earth.

Electroacupuncture for the management of symptom clusters in cancer patients and survivors (EAST).

BACKGROUND: Neuropsychiatric symptoms, comprising cognitive impairment, fatigue, insomnia, depression, and anxiety, are prevalent and may co-occur during and after chemotherapy treatment for cancer. Electroacupuncture (EA), which involves mild electrical stimulation with acupuncture, holds great potential in addressing the management of individual symptoms. However, there is a lack of studies evaluating if EA can manage concurrent neuropsychiatric symptoms in cancer (i.e., symptom cluster). Hence, we designed a trial to evaluate the efficacy, safety, and feasibility of administering EA as an intervention to mitigate neuropsychiatric symptom clusters amongst cancer patients and survivors. METHODS: The EAST study is a randomized, sham-controlled, patient- and assessor-blinded clinical trial. Sixty-four cancer patients and survivors with complaints of one or more neuropsychiatric symptom(s) in the seven days prior to enrollment are recruited from the University of California Irvine (UCI) and Children's Hospital of Orange County (CHOC). Individuals with needle phobia, metastases, bleeding disorders, electronic implants, epilepsy, exposure to acupuncture in the three months prior to enrollment, and who are breastfeeding, pregnant, or planning to get pregnant during the duration of the study will be excluded. Screening for metal fragments and claustrophobia are performed prior to the optional neuroimaging procedures. Recruited patients will be randomized (1:1) in random blocks of four or six to receive either ten weekly verum EA (treatment arm, vEA) or weekly sham EA (control arm, sEA) treatment visits with a follow-up appointment four to twelve weeks after their last treatment visit. The treatment arm will receive EA at 13 acupuncture points (acupoints) chosen for their therapeutic effects, while the control arm receives minimal EA at 7 non-disease-related acupoints. Questionnaires and cognitive assessments are administered, and blood drawn to assess changes in symptom clusters and biomarkers, respectively. CONCLUSION: The EAST study can provide insight into the efficacy of EA, an integrative medicine modality, in the management of cancer symptom clusters in routine clinical practice. TRIAL REGISTRATION: This trial is registered with clinicaltrials.gov NCT05283577.

CkREV regulates xylem vessel development in Caragana korshinskii in response to drought.

Drought stress poses severe threat to the development and even the survival status of plants. Plants utilize various methods responding to drought, among which the forming of more well-developed xylem in leaf vein in woody plants deserves our attention. Herein, we report a transcription factor CkREV from HD-ZIP III family in Caragana korshinskii, which possesses significant functions in drought response by regulating xylem vessel development in leaf vein. Research reveal that in C. korshinskii the expression level of CkREV located in xylem vessel and adjacent cells will increase as the level of drought intensifies, and can directly induce the expression of CkLAX3, CkVND6, CkVND7, and CkPAL4 by binding to their promoter regions. In Arabidopsis thaliana, CkREV senses changes in drought stress signals and bidirectionally regulates the expression of related genes to control auxin polar transport, vessel differentiation, and synthesis of cell wall deposits, thereby significantly enhancing plant drought tolerance. In conclusion, our findings offer a novel understanding of the regulation of CkREV, a determinant of leaf adaxial side, on the secondary development of xylem vessels in leaf vein to enhance stress tolerance in woody plants.

Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived from xCas9.

The recognition of protospacer adjacent motif (PAM) is a key factor for the CRISPR (i.e. clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (Cas9) system to distinguish foreign DNAs from the host genome, and also significantly restricts the targeting scope of the system during genome-editing applications. Structurally, the PAM interacting (PI) domain, which usually is located in the C-terminus of Cas9 proteins, directly binds to PAM and plays a key role in determining the recognition specificity. However, several lines of evidence showed that other regions of Cas9 protein beyond the PI domain might also play roles in PAM interaction. Here, we constructed a mosaic SpCas9 protein (xCas9-NG) by fusing the PI domain of SpCas9 PAM variant, Cas9-NG with the non-PI fragment of xCas9 protein that contains multiple amino acid substitutions. We found that non-PI fragment of xCas9 expanded PAM recognition of the Cas9-NG PI domain. In addition, xCas9-NG showed an improved editing efficiency in the majority of targets harboring xCas9 and Cas9-NG PAMs. Importantly, this finding was also successfully extended to other Cas9 variants, including SpRY and the non-G SpCas9 series. Together, our work expands the target scope of SpCas9 editing system and demonstrates the notion that the non-PI domain fragment plays an important role in PAM restriction.