The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided...The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease,Cas12i3.As a result,we developed Cas-SF01,a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells.Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases.Compared to natural Cas12i3,Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs.In addition,we identified an amino acid substitution,D876R,that markedly reduced the off-target effect while maintaining high on-target activity,leading to the development of CasSF01^(HiFi)(high-fidelity Cas-SF01).Finally,we show that Cas-SF01 has high gene editing activities in mice and plants.Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.展开更多
Background and Aims:Hepatocellular carcinoma(HCC)is the most common primary liver cancer and the incidence and mortality rates are increasing.Given the limited treatments of HCC and promising application of immunother...Background and Aims:Hepatocellular carcinoma(HCC)is the most common primary liver cancer and the incidence and mortality rates are increasing.Given the limited treatments of HCC and promising application of immunotherapy for cancer,we aimed to identify an immune-related prognostic signature that can predict overall survival(OS)rates and immunotherapy response in HCC.Methods:The initial signature development was conducted using a training dataset from the Cancer Genome Atlas followed by independent internal and external validations from that resource and the Gene Expression Omnibus.A signature based nomogram was generated using multivariate Cox regression analysis.The associations of signature score with tumor immune phenotype and response to immunotherapy were analyzed using single-sample gene set enrichment analysis and tumor immune dysfunction and exclusion algorithm.A cohort from Zhongshan Hospital was employed to verify the pre dictive robustness of the signature regarding prognostic risk and immunotherapy response.Results:The prognostic signature,IGS_(HCC),consisting of 22 immune-related genes,had independent prognostic ability,with training and validation cohorts.Also,IGS_(HCC)stratified HCC patients with different outcomes in subgroups.The prognostic accuracy of IGS_(HCC)was better than three reported prognostic signatures.The IGS_(HCC)-based nomogram had high accuracy and significant clinical benefits in predicting 3-and 5-year OS.IGS_(HCC)reflected distinct immunosuppressive phenotypes in low-and high-score groups.Patients with low IGS_(HCC)scores were more likely than those with high scores to benefit from immunotherapy.Conclusions:IGS_(HCC)predicted HCC prognosis and response to immunotherapy,and contributed to individualized clinical management.展开更多
Enhancers activate transcription in a distance-,orientation-,and position-independent manner,which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the ...Enhancers activate transcription in a distance-,orientation-,and position-independent manner,which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes,especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40%of identified enhancers car-ried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks,H3K4me3 and/or H3K27ac,which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes,which overlap poorly with STARR-seq enhancers. In summary,we quantitatively identified enhancers by functional analysis in the genome of rice,an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.展开更多
基金This work was supported by Shandong BellaGen Biotechnology,and by a grant from the National Natural Science Foundation of China(32188102 to J.-K.Z.).
文摘The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease,Cas12i3.As a result,we developed Cas-SF01,a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells.Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases.Compared to natural Cas12i3,Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs.In addition,we identified an amino acid substitution,D876R,that markedly reduced the off-target effect while maintaining high on-target activity,leading to the development of CasSF01^(HiFi)(high-fidelity Cas-SF01).Finally,we show that Cas-SF01 has high gene editing activities in mice and plants.Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.
基金supported by the National Natural Science Foun-dation of China(82103521,82073208)the Shanghai Sailing Program(21YF1407500)+2 种基金the China Postdoctoral Science Foun-dation(2021M690674)the Special Foundation for Science and Technology Basic Research Program(2019FY101103)the Shanghai Shen Kang Hospital Development Center new frontier technology joint project(SHDC12021109).
文摘Background and Aims:Hepatocellular carcinoma(HCC)is the most common primary liver cancer and the incidence and mortality rates are increasing.Given the limited treatments of HCC and promising application of immunotherapy for cancer,we aimed to identify an immune-related prognostic signature that can predict overall survival(OS)rates and immunotherapy response in HCC.Methods:The initial signature development was conducted using a training dataset from the Cancer Genome Atlas followed by independent internal and external validations from that resource and the Gene Expression Omnibus.A signature based nomogram was generated using multivariate Cox regression analysis.The associations of signature score with tumor immune phenotype and response to immunotherapy were analyzed using single-sample gene set enrichment analysis and tumor immune dysfunction and exclusion algorithm.A cohort from Zhongshan Hospital was employed to verify the pre dictive robustness of the signature regarding prognostic risk and immunotherapy response.Results:The prognostic signature,IGS_(HCC),consisting of 22 immune-related genes,had independent prognostic ability,with training and validation cohorts.Also,IGS_(HCC)stratified HCC patients with different outcomes in subgroups.The prognostic accuracy of IGS_(HCC)was better than three reported prognostic signatures.The IGS_(HCC)-based nomogram had high accuracy and significant clinical benefits in predicting 3-and 5-year OS.IGS_(HCC)reflected distinct immunosuppressive phenotypes in low-and high-score groups.Patients with low IGS_(HCC)scores were more likely than those with high scores to benefit from immunotherapy.Conclusions:IGS_(HCC)predicted HCC prognosis and response to immunotherapy,and contributed to individualized clinical management.
基金the financial support from the National Natural Science Foundation of China (Grant Nos.31571347 to CH and 31771430 to LL)Guangdong Science and Technology Department, China (Grant No. 2016A030313642 to CH)+2 种基金Shenzhen Science and Technology Innovation Commission, China (Grant No. JCYJ20150529152146478 to CH)Huazhong Agricultural University Scientific & Technological Self-innovation Foundation, China to LLthe Thousand Youth Talents Plan of China to CH
文摘Enhancers activate transcription in a distance-,orientation-,and position-independent manner,which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes,especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40%of identified enhancers car-ried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks,H3K4me3 and/or H3K27ac,which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes,which overlap poorly with STARR-seq enhancers. In summary,we quantitatively identified enhancers by functional analysis in the genome of rice,an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.