Nanopore Sequencing-Mediated CRISPR Activation Toolkit Optimization And Custom Pool Construction And Screening

CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)has evolved from an initial gene editing tool to a gRNA(guide RNA)targeted gene editing technology system with multiple gene editing functions,such as pilot editing,base editing,gene activation,gene disruption and gene knockout,etc.Recent advancements in DNA synthesis technology have made it easier to synthesize custom gRNA libraries,which can be combined with CRISPR to conduct high-throughput research on specific genes.This technology has great potential for modifying microbial cell factories and studying cellular metabolic networks.However,there is currently a lack of quality control tools for library construction and editing tools need to be improved.To address these issues,our study aims to use third-generation nanopore sequencing technology to monitor library quality and optimize the construction method of customized gRNA plasmid libraries.With these improvements,we hope to create a more efficient CRISPR activation tool.First,for the construction of gRNA custom libraries,we established a highthroughput characterization method based on nanopore sequencing and evaluated existing custom library construction techniques such as Golden Gate and Gibson Assembly,and the results showed that Golden Gate and Gibson Assembly.The results showed that the gRNA ratios of Golden Gate and Gibson Assembly were highly discrete with standard deviations of 6.25%and 3.02%,respectively.To address the highly discrete number of gRNAs in the library,we improved the homogeneity of the library with a PCR-mediated non-amplification method,and the standard deviation was reduced to 2.01%,which enabled the abundance of each gRNA to be guaranteed.Second,for the modification of the dCas12a-based activation tool,we tested the activation structural domains of multiple transcription factors and used gRNA libraries to rapidly determine the optimal combination of activation domains and gRNAs.Combining nanopore sequencing and high-throughput screening to improve the efficiency and accuracy of the screening,the results showed that the optimal activation domain MED2 and gRNA could significantly increase the transcriptional activity of multiple promoters of different intensities,and the expression efficiency of PARO4,PCYC1 and PTEE1 was increased by 10-fold,7-fold and 2-fold,respectively,using red fluorescent protein as a reporter gene.Subsequently,we ordered a customized library with 99 gRNAs that target 35 genes in the central metabolism.Our goal was to find gene targets that enhance ethanol utilization in yeast.Using the model microorganism Saccharomyces cerevisiae,we demonstrated our screening method based on the fold change in gRNA abundance under growth pressure.We were able to identify the most significantly enriched targets:ACH1-3,ADH4-4,and ADH2-2.After validation,we found that the strains with ACH1,ADH2,and ADH4 activated had increased OD600 values(compared to the control strain)of 2.4-fold,1.9-fold,and 1.5-fold,respectively,after 120 hours of growth in YNB medium.Finally,we used a combination of flow cytometry and a tyrosine biosensor based on betaxanthine accumulation,along with genome-wide gRNA libraries,to conduct high throughput screening of yeast endogenous genes for up-regulation,downregulation,and knockout on a genome-wide scale.Through this approach,we were able to identify multiple candidate gene targets that can be used to create engineered Saccharomycete cerevisiae cells capable of efficiently synthesizing tyrosine derivatives.Our results revealed that PMR1 is responsible for regulating the internal mcroenvironment of cells,such as pH,Ca2+ concentration and signaling pathways.We also found that down-regulating SIT4,as well as up-regulating NAG1 and PIK1,can further increase the accumulation of betaxanthine in yeast after removing negative tyrosine feedback.This study can serve as an important reference and support for the further development of tyrosine metabolism engineering.Overall,this study utilized third-generation nanopore sequencing technology and introduced a custom library construction and quality control method.We also improved CRISPR activation tools in yeast to address the issue of low activation efficiency.Through our work,we successfully identified targets that promote efficient ethanol utilization in yeast.Additionally,we conducted a comprehensive genome-wide screen for gene targets that increase tyrosine production,leading to the discovery of new targets for metabolic engineering modifications to further enhance tyrosine production.