Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library

Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.

Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.

Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.

Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens

Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.

Boosting genome editing in plants with single transcript unit surrogate reporter systems

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement.However,the challenge of low editing activity complicates the identification of editing events.In this study,we introduce multiple single transcript unit surrogate reporter(STU-SR)systems to enhance the selection of genome-edited plants.These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes,establishing a direct link between reporter gene editing activity and that of endogenous genes.Various strategies are used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA systems.STU-SR-base editor systems leverage base editing to reinstate the start codon,enriching C-to-T and A-to-G base editing events.Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice,encompassing Cas9 nuclease-based targeted mutagenesis,cytosine base editing,and adenine base editing.The systems exhibit compatibility with Cas9 variants,such as the PAM-less SpRY,and are shown to boost genome editing in Brassica oleracea,a dicot vegetable crop.In summary,we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.