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<p>Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from <i>Streptococcus pyogenes</i> complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.</p>

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Design principle of successful genome editing applications using CRISPR-based toolkits

  • 期刊论文

  • 发表日期：

    2025年7月1日

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  • Juhi Sharma,
  • Rajesh Biswas,
  • Prashant Khare

摘要

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from Streptococcus pyogenes complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.