CRISPR-CBEI: a Designing and Analyzing Tool Kit for Cytosine Base Editor-Mediated Gene Inactivation

• Published in: mSystems 2020-10, Vol.5 (5)
• Main Authors: Yu, Haopeng, Wu, Zhaowei, Chen, Xiangdan, Ji, Quanjiang, Tao, Shiheng
• Format: Article
• Language: English
• Subjects: base editing; Bioinformatics; Codons; CRISPR; Cytosine; cytosine base editor; Deoxyribonucleic acid; Design; DNA; DNA damage; gene inactivation; Genome editing; Genomes; Molecular Biology and Physiology; Mutagenesis; Mutation; Nuclease; Nucleotide sequence; Open reading frames; Prokaryotes; Recombination; Species; Thymidine; Transcription; Transcription activator-like effector nucleases; Trinucleotide repeats; Zinc finger proteins
• ISSN: 2379-5077; 2379-5077
• DOI: 10.1128/mSystems.00350-20

Life science has been in pursuit of precise and efficient genome editing in living cells since the very beginning of the first restriction cloning attempt. The introduction of RNA-guided CRISPR-associated (Cas) nucleases contributed to this ultimate goal through their ability to deliver a double-strand break (DSB) to a precise target location in various species, obsoleting the preceding editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The derivative technology, base editing, combines the catalytically inactivated Cas nuclease and nucleotide deaminase and mediates the genetic modifications at single-nucleotide precision without introducing a DSB. Moreover, the cytosine base editors (CBEs) are able to transform multiple codons into stop codons, rapidly inactivating a gene of interest and enabling loss-of-function study in some recombination-deficient species. Here, we present the CRISPR-CBEI tool kit to assist the design of sgRNAs for CBE-mediated gene inactivation. Base editing is a promising technique, allowing precise single-base mutagenesis in genomes without double-strand DNA breaks or donor templates. Cytosine base editors (CBEs) convert cytosine to thymidine. In particular, CBEs can transform four codons, CAA, CAG, CGA, and TGG, into stop codons, providing a new means to rapidly inactivate a gene of interest and enabling loss-of-function study in recombination-deficient species and the construction of gene-inactivation libraries. However, designing single guide RNAs (sgRNAs) for gene inactivation is more complicated and more restricted in applicability than using the lustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CRISPR/Cas9) system only, especially for researchers who do not specialize in the bioinformatics skills needed to design and evaluate sgRNAs. Here, we present a new user-friendly designing tool kit, namely, CRISPR-CBEI ( c ytosine b ase e ditor-mediated gene i nactivation), including a Web tool and a command-line tool. The Web tool is dedicated to the design of sgRNAs for CBE-mediated gene inactivation and integrates various functions, including open reading frame (ORF) identification, CBE customization, sgRNA designing, summarizing, and front-end off-target searching against user-defined unlimited-file-size local genome files without the necessity of uploading to the server. The command-line version serves the same purpose but for a larger