Materials and Methods

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

MATERIALS AND METHODSThe CRISPR-CBEI Web tool. The Web-based CRISPR-CBEI tool is freely accessible at https://taolab.nwafu.edu.cn/crisprcbei/ or https://atlasbioinfo.github.io/CRISPR-CBEI/. It adopts HTML5 as the framework, CSS3 for the layout, JavaScript for the calculation process, and the Web Workers of HTML5 for loading local files. The built-in genetic codes and values in the ORF identification function are based on ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). JavaScript (JS) is used to perform the translation of the user-customized base editor parameter into a regular expression. Then, a regular expression-matching algorithm is initiated to search for the potential spacers which perfectly match the regular expression within the user-selected ORF. Subsequently, the program judges whether a deoxycytidine of the four editable codons, CAA, CAG, CGA, and TGG, is present in the defined editing window and whether the editing resulted in a premature stop codon. Finally, the program presents all potential CBEI spacers within the user-defined search region of the target ORF.Front-end off-target searching. HTML Web Workers were adopted in CRISPR-CBEI to enable local data processing without the necessity of uploading large genomic files to the server. The front-end off-target searching first splices the spacer into “mismatch + 1” fragments, termed anchors. A sequence alignment is then started to match the genome with these anchors. If the anchor matches, the program then determines whether its upstream sequence or downstream sequence contains a PAM of the selected CBE. After the PAM determination, the mismatch value representing the remaining part on the spacer is compared with the set value. Spacers that have a lower mismatch value than the pre-set value are considered potential off-target sites. The efficiency of the search process depends on CPU performance, genome size, and the mismatch value. The front-end off-target searching algorithm supports the use of large genome files by breaking the genome file into 50-Mb segments. Accordingly, 100-nt sequences of the upstream and downstream regions flanking the break site are extracted as new segments for the off-target searching process to avoid neglecting the off-target site near the break site.The “autocbei” Python-based command-line tool. The source code and detailed instructions are deposited in Github for free access (see below). This command-line tool is written in Python 3 and requires “biopython” and “matplotlib” packages for running. The “autocbei” command-line tool has been uploaded to PyPI and Anaconda Cloud for easy installation with “pip” and “conda.” More installation and usage information is available on the corresponding Github (https://github.com/atlasbioinfo/CRISPR-CBEI/blob/master/autocbei/README.md), PyPI (https://pypi.org/project/autocbei/), and Anaconda (https://anaconda.org/atlasbioinfo/autocbei) Web pages. We also set up “TravisCI” for continuous integration testing for “autocbei.” CDS data for the 12 model species used in the analyses were obtained from the Ensembl database.Support and tutorials. The CRISPR-CBEI tool kit includes two applications, the Web tool and the command-line tool (“autocbei”), both of which contain detailed instructions. An introduction to the Web version of CRISPR-CBEI can be found on the “Help” page of the website, where we provided a downloadable complete user manual in a PDF file. Detailed information regarding the installation and usage of the “autocbei” tool is provided in the Github “README” file and can also be obtained through the “-h” parameter. If users have problems using both versions of the CRISPR-CBEI tool kit, they can submit a Github issue or contact the project administrator by email. Contact details can be found on the CRISPR-CBE webpage or Github project homepage.Data availability. We provide the source code for the Web-based CRISPR-CBEI tool on Github (https://github.com/atlasbioinfo/CRISPR-CBEI/). The source code and detailed instructions are deposited in Github for free access (https://github.com/atlasbioinfo/CRISPR-CBEI/tree/master/autocbei).

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

Abstract

We provide the source code for the Web-based CRISPR-CBEI tool on Github (https://github.com/atlasbioinfo/CRISPR-CBEI/). The source code and detailed instructions are deposited in Github for free access (https://github.com/atlasbioinfo/CRISPR-CBEI/tree/master/autocbei).


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