Bioscience Evidence 2024, Vol.14, No.3, 131-142 http://bioscipublisher.com/index.php/be 135 Figure 2 Applications of multiplexed CRISPR–Cas technologies (Adopted from McCarty et al., 2020) Image caption: a Multiple gRNAs can be expressed, together with dCas9, to build complex logic circuits, including wired NOR gates, in which upstream gRNAs regulate the expression of downstream sgRNAs. Logic circuits can be used to produce a simple output signal, like GFP, or can be interfaced into cellular pathways to control phenotypes or behaviors. b Cas13a orthologs can be used to detect multiple viral pathogens at once. The viruses are lysed, their genomes are amplified, and the amplified RNA is then used as the input for Cas13a-based biosensors. Upon recognition of an RNA target, Cas13a collaterally cleaves nearby transcripts, a characteristic that can be exploited to release orthogonal, fluorescent outputs from ssRNA reporters. c Multiplexed gRNAs enable combinatorial mapping of genotype to phenotype. Pairs of gRNAs, each with a unique barcode, are programmed to target different genes involved in a known pathway or cellular process. These gRNA:barcode pairs are transformed into Cas9- expressing cells, and the barcodes of each cell in a population are sequenced to determine which gRNA pair each cell received. By measuring the frequency of the barcodes over multiple conditions, combinations of genes that modulate a given phenotype can be inferred. d Multi-event recording enables multiple signals to be detected and recorded in the genome of living cells. One gRNA is used to “write” each detected signal. Event recorders commonly use base editors and gRNAs that target a pre-defined locus, and recordings can be read out by sequencing the targeted loci. e Multiplexed CRISPR–Cas enables specific genomic rearrangements or modifications, including indels (which are produced by error-prone, non-homologous end joining) and insertions (via homology-directed repair, where donor DNA contains homology arms to the double-strand break), for rapid strain engineering. f Multiplexed CRISPR–Cas technologies can be used to perturb numerous parts of a pathway simultaneously, thus redirecting flux and enhancing the production of a desired compound. CRISPRi, CRISPRa, and editing of DNA can be achieved simultaneously, simply by expressing orthogonal dCas:gRNA pairs (one for activation and another for repression), together with Cas12a or Cas9 for editing (Adopted from McCarty et al., 2020) 5 Case Studies and Applications 5.1 Enhanced enzymes for biofuel production: lignocellulosic biomass breakdown The application of synthetic biology in directed evolution has significantly advanced the efficiency of enzymes used in the breakdown of lignocellulosic biomass for biofuel production. For instance, genetically engineered proteins have been tailored to improve biomass conversion, enhancing the catalytic performance of enzymes involved in lignocellulosic polymer degradation (Ribeiro et al., 2019). Additionally, extremophilic bacteria have been utilized to provide robust enzymes capable of functioning under harsh industrial conditions, thereby improving the efficiency of biofuel production processes (Zhu et al., 2020). The use of nano-biocatalysts, such as xylanase immobilized on mesoporous silica nanoparticles, has also shown to enhance the stability and recyclability of enzymes, leading to improved degradation of lignocellulosic agro-waste (Ariaeenejad et al., 2020). Metagenomic techniques have further facilitated the discovery of novel enzymes from microbial communities,
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