Contribution | Gifu

Developing a multi-targetable sgRNA designing tool PAMConAligner

From the discussion with Dr. Akio Suzuki and Dr. Takashi Niwa of the Department of Pharmacy, Gifu University Hospital, the following two points became clear. The emergence of drug-resistant bacteria will limit the treatment if antibiotic therapy is continued as it is now. Knocking out the gene for drug-resistant bacteria is an effective way to continue antibiotic therapy.

To solve the above problems, we decided to use the CRISPR/Cas system, a common gene knockout method, for our project. Typically, a single sgRNA is designed for a single target sequence selected from the target gene and knocked out using the CRISPR/Cas system. In order to more efficiently knock out multiple antibiotic resistance gene variants, we needed to design sgRNAs that could more efficiently recognize and respond to multiple variants. Therefore, we focused on [1]CRISPR MultiTargeter (Multi Targeter), a sgRNA design software tool that can design sgRNA sequences that recognize all input sequences by inputting multiple candidate target sequences. We identified two weaknesses in the use of MultiTargeter and developed a new improved software, PAMConAligner, to make it usable.

Weak points of MultiTargeter

・MultiTargeter designs gRNAs only by aligning input sequences and obtaining consensus sequences and does not consider evolutionary information.

・MultiTargeter can only design gRNAs that match all input sequences or a gRNA that matches only arbitrary sequences.

Improvements in PAMConAligner

・Enables evolutionary information to be taken into account when designing gRNAs.

・Allows the generation of gRNAs that maximize the number of matching sequences, even if not all gRNAs match all sequences.

Our PAMConAligner software is advantageous for the discovery and design of sgRNAs from multiple input candidate target sequences, and we believe that it can contribute to the iGEM community and the field of molecular biology through efficient and rapid sgRNA design. Furthermore, as addressed in the project, gRNAs designed with PAMConAligner are expected to have applications in the medical field. As an example, it may be possible to knock out the coagulase gene retained by methicillin-resistant Staphylococcus aureus (MRSA), which is currently a problem in the medical field.

Since the PAMConAligner software was developed with the goal of designing gRNA, it can also be used to design ssDNA/ssRNA targeting homologues. In the future, it is expected to be used in the field of synthetic biology for the design of siRNAs used in RNA interference (RNAi).

MultiTargeter and other tools for designing sgRNAs from common sequences are also described by our team on page BBa_J428062.

Reference

[1] Sergey V. Prykhozhij et al.(2015): CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences,PLOS ONE 10(9): e0138634. *Linked Here"