Construction of Efficient Platform Escherichia coli Strains for Polyhydroxyalkanoate Production by Engineering Branched Pathway
Hye‐Rim JungSuyeon YangYu‐Mi MoonTae‐Rim ChoiHun‐Suk SongShashi Kant BhatiaRanjit GuravEun-Jung KimByung‐Gee KimYung‐Hun Yang
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Abstract:
Polyhydroxyalkanoate (PHA) is a potential substitute for petroleum-based plastics and can be produced by many microorganisms, including recombinant Escherichia coli. For efficient conversion of substrates and maximum PHA production, we performed multiple engineering of branched pathways in E. coli. We deleted four genes (pflb, ldhA, adhE, and fnr), which contributed to the formation of byproducts, using the CRISPR/Cas9 system and overexpressed pntAB, which catalyzes the interconversion of NADH and NADPH. The constructed strain, HR002, showed accumulation of acetyl-CoA and decreased levels of byproducts, resulting in dramatic increases in cell growth and PHA content. Thus, we demonstrated the effects of multiple engineering for redirecting carbon flux into PHA production without any concerns regarding simultaneous deletion.Keywords:
Metabolic Engineering
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a powerful gene editing tool originated from prokaryotes. The modern CRISPR/Cas9 system allows high-throughput genetic screening to be carried out in vitro and in vivo. The high efficacy and flexibility of CRISPR/Cas9 system allows identification of hepatocellular carcinoma (HCC) related genes in the past decades. Numerous efforts have been applied in the past to improve this system, such as off-target improvement, gRNA efficiency enhancement, and modified Cas9 nuclease for performing visionary base editor screens and epigenetic screens. With these merits, the CRISPR/Cas9 offers tremendous opportunities in various biomedical research and clinical application. Recently, the use of the CRISPR/Cas9 system has also been combined with different technologies, including single-cell sequencing and machine learning, to further understand HCC pathogenesis and explore its utility in gene therapy. This review provides a summary of HCC carcinogenesis CRISPR/Cas9 screens conducted in recent years with different genetic contexts, epidemiological backgrounds, and progression of HCC. Furthermore, this review also provides insight in the CRISPR/Cas9 potentials, current obstacles, and improvement of this system for its future utility in cocktail therapies.
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CRISPR/Cas9 genome editing technology is the frontier of life science research. They have been used to cure human genetic diseases, achieve cell personalized treatment, develop new drugs, and improve the genetic characteristics of crops and other fields. This system relies on the enzyme Cas9 cutting target DNA (on target) under the guidance of sgRNA, but it can also cut non-target sites, which results in offtarget effects, thus causing uncontrollable mutations. The risk of off-target effect in CRISPR technology is the main limiting factor that affects the widespread application of CRISPR technology. How to evaluate and reduce the off-target effect is the urgent problem to be solved. In this work, we build up a model that can predict the score of being off-target. Through comparison with the complete genome of the target and precise mathematics that calculate the potential risk of being off-target, we optimize the sgRNA, which is capable of reducing the off-target effect. The result has proven that we can efficiently and quickly identify and screen the best editing target sites with our model. The CRISPR/Cas9 system, not even being perfected yet, has already demonstrated its potential in the field of genome editing. Hopefully through our model, the CRISPR/Cas9 system can quickly apply to more branches in life science and cure those diseases that have been previously incurable.
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Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to singlestranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology. [BMB Reports 2023; 56(2): 102-107].
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Abstract The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a ( p lasmid of synthetic CRI SPR-coded RNA target sequence-equipped donor plasmid- m ediated ge ne t argeting via Cas 9 and Cas 12a ), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.
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CRISPR/Cas9 gene editing technology has revolutionized genetic engineering. However, the nuclear dynamics of Cas9 in eukaryotic cells, particularly in the model organism
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The clustered regulatory interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system is the part of the prokaryotic immune system, which could recognize and delete the exogenous sequences originated from virus or plasmid. Based on its mechanism, CRISPR-Cas9 system was developed into the new generation of gene editing tool. Compared to the existed technologies such as ES targeting, ZFN or TALEN, CRISPR-Cas9 system is a more efficient, economical and promising approach to manipulate the genome. In this review, we summarize the research progress about CRISPR-Cas9 technology, especially the latest applications in gene therapy studies of human diseases.CRISPR-Cas9 系统是细菌在与噬菌体抗争的进化过程中产生的一种抵御外源DNA 入侵的机制，能有效识别并剪切外源DNA。基于其识别切除外源DNA 的原理，CRISPR-Cas9 系统被开发成为新一代基因编辑工具。与ES 打靶、ZFN、TALEN 等技术途径相比，CRISPR-Cas9 系统操作简便、效率高、成本低，有着极其广阔的应用前景。本文整理了近年内有关CRISPR-Cas9 系统的最新文献报道，对该系统工作原理以及针对基因治疗的研究进展进行综述。.
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Abstract Large deletions and genomic re‐arrangements are increasingly recognized as common products of double‐strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats ‐ CRISPR associated protein 9 (CRISPR/Cas9) on‐target sites. Together with well‐known off‐target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9‐editing is necessary to ensure validity of observed phenotypes in Cas9‐edited cell‐lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide‐RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off‐targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on‐ and off‐target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.
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