Breakthrough in CRISPR/Cas system: Current and future directions and challenges

Ali, Ahmad; Zafar, Muhammad Mubashar; Farooq, Zunaira; Ahmed, Syed Riaz; Ijaz, Aqsa; Anwar, Zunaira; Abbas, Huma; Tariq, Muhammad Sayyam; Tariq, Hala; Mustafa, Mahwish; Bajwa, Mubasher Hussain; Shaukat, Fiza; Razzaq, Abdul; Maozhi, Ren

doi:10.1002/biot.202200642

Cited by 6 publications

(5 citation statements)

References 240 publications

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“…The CRISPR/Cas9 systems have undergone significant advancements [ 26 ] and have been extensively employed in various filamentous fungi owing to their rapidity and efficiency. [ 5,8,10,12,16,22,27 ] The development of efficient and robust genetic operating systems in nonmodel filamentous fungi remains unknown challenges.…”

Section: Discussionmentioning

confidence: 99%

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi

Huang,

Li,

Song

et al. 2024

Biotechnology Journal

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Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50 bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3‐hydroxy‐3‐methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid‐based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single‐plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.

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Section: Discussionmentioning

confidence: 99%

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi

Huang,

Li,

Song

et al. 2024

Biotechnology Journal

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“…Recently existing base editors, encompassing adenine base editors (ABEs) and cytosine base editors (CBEs), have been engineered by fusing the inactive Cas9 (dCas9) or Cas9 nickase (D10A) (nCas9) with adenine deaminase or cytidine deaminase, respectively ( Komor et al, 2016 ; Gaudelli et al, 2017 ). In this manner, single-point mutations such as the conversion of A*T to G*C or C*G to T*A in crop plants can be generated using ABEs or CBEs ( Bharat et al, 2020 ; Ali et al, 2023 ; Khan et al, 2023 ). So far, base editing has been effectively used to target several important traits in various crops like maize, cotton, Arabidopsis thaliana , rice and others.…”

Section: Revolutionizing Genetics: Genome Editing and Beyondmentioning

confidence: 99%

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus

Nadeem,

Riaz Ahmed,

Luqman

et al. 2024

Front. Genet.

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Cotton (Gossypium hirsutum L.) is a significant fiber crop. Being a major contributor to the textile industry requires continuous care and attention. Cotton is subjected to various biotic and abiotic constraints. Among these, biotic factors including cotton leaf curl virus (CLCuV) are dominant. CLCuV is a notorious disease of cotton and is acquired, carried, and transmitted by the whitefly (Bemisia tabaci). A cotton plant affected with CLCuV may show a wide range of symptoms such as yellowing of leaves, thickening of veins, upward or downward curling, formation of enations, and stunted growth. Though there are many efforts to protect the crop from CLCuV, long-term results are not yet obtained as CLCuV strains are capable of mutating and overcoming plant resistance. However, systemic-induced resistance using a gene-based approach remained effective until new virulent strains of CLCuV (like Cotton Leaf Curl Burewala Virus and others) came into existence. Disease control by biological means and the development of CLCuV-resistant cotton varieties are in progress. In this review, we first discussed in detail the evolution of cotton and CLCuV strains, the transmission mechanism of CLCuV, the genetic architecture of CLCuV vectors, and the use of pathogen and nonpathogen-based approaches to control CLCuD. Next, we delineate the uses of cutting-edge technologies like genome editing (with a special focus on CRISPR-Cas), next-generation technologies, and their application in cotton genomics and speed breeding to develop CLCuD resistant cotton germplasm in a short time. Finally, we delve into the current obstacles related to cotton genome editing and explore forthcoming pathways for enhancing precision in genome editing through the utilization of advanced genome editing technologies. These endeavors aim to enhance cotton’s resilience against CLCuD.

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“…[1,2] The writing, reading, and editing of random DNA sequences are vital important for most fields of modern biology. [3][4][5] The phosphoramidite method for chemical DNA synthesis was developed in the 1980s, boosting the field of DNA synthesis in vitro. [6] In the following 40 years, various methods were developed to improve the synthesis speed and accuracy, and thus meet the demands of the rapid development of life sciences.…”

Section: Introductionmentioning

confidence: 99%

Recent advances in the synthesis of single‐stranded DNA in vitro

Li,

Tan,

Jia

et al. 2024

Biotechnology Journal

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Single‐stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid‐phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase‐based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.

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Breakthrough in CRISPR/Cas system: Current and future directions and challenges

Cited by 6 publications

References 240 publications

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus

Recent advances in the synthesis of single‐stranded DNA in vitro

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