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The application of clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) technology in the genetic modification of Yarrowia lipolytica is challenged by low efficiency and low throughput. Here, a highly efficient CRISPR-iCas9 (with D147Y and P411T mutants) genetic manipulation tool was established for Y. lipolytica, which was further utilized to integrate carotene synthetic key genes and significantly improve the target product yield. First, CRISPR-iCas9 could shorten the time of genetic modification and enable the rapid knockout of nonsense suppressors. iCas9 can lead to more than 98% knockout efficiency for NHEJ-mediated repair after optimal target disruption of a single gene, 100% knockout efficiency for a single gene-guided version, and more than 80% knockout efficiency for multiple genes simultaneously in Y. lipolytica. Subsequently, this technology allowed for rapid one-step integration of large fragments (up to 9902-bp) of genes into chromosomes. Finally, YL-ABTG and YL-ABTG2Z were further constructed through CRISPR-iCas9 integration of key genes in a one-step process, resulting in a maximum β-carotene and zeaxanthin content of 3.12 mg/g and 2.33 mg/g dry cell weight, respectively. Therefore, CRISPR-iCas9 technology provides a feasible approach to genetic modification for efficient biosynthesis of biological compounds in Y. lipolytica. Key points: • iCas9 with D147Y and P411T mutants improved the CRISPR efficiency in Y. lipolytica. • CRISPR-iCas9 achieved efficient gene knockout and integration in Y. lipolytica. • CRISPR-iCas9 rapidly modified Y. lipolytica for carotenoid bioproduction. [ABSTRACT FROM AUTHOR]

Malignant tumors pose a serious threat to human health and have high fatality rates. Conventional clinical anti-tumor treatment is mainly based on traditional surgery, chemotherapy, radiotherapy, and interventional therapy, and even though these treatment methods are constantly updated, a satisfactory efficacy is yet to be obtained. Therefore, research on novel cancer treatments is being actively pursued. We review the classification of gene therapies of malignant tumors and their advantages, as well as the development of gene editing techniques. We further reveal the nano-drug delivery carrier effect in improving the efficiency of gene editing. Finally, we summarize the progress in recent years of gene editing techniques based on nano-drug delivery carriers in the treatment of various malignant tumors, and analyze the prospects of the technique and its restricting factors.

The clustered regularly-interspaced short palindromic repeats (CRISPR) structural family functions as an acquired immune system in prokaryotes. Gene editing techniques have co-opted CRISPR and the associated Cas nucleases to allow for the precise genetic modification of human cells, zebrafish, mice, and other eukaryotes. Indeed, this approach has been used to induce a variety of modifications including directed insertion/deletion (InDel) of bases, gene knock-in, introduction of mutations in both alleles of a target gene, and deletion of small DNA fragments. Thus, CRISPR technology offers a precise molecular tool for directed genome modification with a range of potential applications; further, its high mutation efficiency, simple process, and low cost provide additional advantages over prior editing techniques. This paper will provide an overview of the basic structure and function of the CRISPR gene editing system as well as current and potential applications to research on parasites. [ABSTRACT FROM AUTHOR]

Malignant tumors pose a serious threat to human health and have high fatality rates. Conventional clinical anti-tumor treatment is mainly based on traditional surgery, chemotherapy, radiotherapy, and interventional therapy, and even though these treatment methods are constantly updated, a satisfactory efficacy is yet to be obtained. Therefore, research on novel cancer treatments is being actively pursued. We review the classification of gene therapies of malignant tumors and their advantages, as well as the development of gene editing techniques. We further reveal the nano-drug delivery carrier effect in improving the efficiency of gene editing. Finally, we summarize the progress in recent years of gene editing techniques based on nano-drug delivery carriers in the treatment of various malignant tumors, and analyze the prospects of the technique and its restricting factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xi, Yang, Suo and Sun.)