CRISPRLY

Software

Overview

Our Team developed a web tool that offers an improved user experience that streamlines the gRNA design process. The web-tool helps in detecting PAM sites and designing gRNAs for large DNA sequences for a range of CRISPR reactions involving different Cas Proteins in an efficient manner. As a final result, the web-app produces gRNA sequences targeting multiple PAM sites detected in the DNA sequence that can be readily ordered for different CRISPR experiments. Since iGEM promotes open science practices, all the codes are available on Github, which makes it easier for future iGEM teams who wish to build upon our work and embed it in new workflows

Click here to visit our Web App

Inspiration

Our project involves the detection of the E7 oncogene of the Human Papillomavirus 16. We are using the trans cleavage property of the CRISPR/Cas12a reaction to detect the E7 gene. The gRNAs involved in the reaction are usually 20-24 nucleotide long sequences that bind to specific sites present on the target DNA. The part of the gRNA sequence that is complementary to the target sequence is known as a spacer. In order for Cas proteins to function, it also requires a specific protospacer adjacent motif (PAM) that varies depending on the type of Cas protein used. In the case of our project, our target E7 oncogene is 298 nucleotides long which may have multiple PAM sites resulting in the possibility of multiple suitable gRNAs for detection. The detection of multiple PAM sites in such a large DNA sequence was very cumbersome and time-consuming, and we realized there is a need for a web-based system which could automate this whole process so that the first model could be made bioinformatically. 

Description

Our webtool tool provides an effective computational method to identify unique targets for gRNAs of the CRISPR/Cas system and design specific gRNAs for the same based on the Cas protein used. 

Our workflow as mentioned below is effortless and straightforward:

  • 1. Upload the file containing the target DNA sequence in its FASTA format or enter the DNA sequence
  • 2. Choose the appropriate Cas protein to be used from the given drop-down menu
  • 3. Results will be displayed in the form of gRNA sequences designed for corresponding PAM sites detected in the target DNA sequence


We have uploaded all of our source code on our github. You can easily access the codes from there and it will also help any future teams to further build upon our work.

Features

Multiple CRISPR/Cas systems

gRNAs can be designed for multiple CRISPR systems in a single web-tool. Most web-tools available only design gRNA for CRISPR/Cas9 systems.

Improved time complexity

Aho-Corasick Algorithm used for the tool significantly improves the time complexity of the string search

Custom PAM site detection

An option to input custom PAM sites can design gRNAs for modified Cas protein systems

Algorithms

The Aho-Corasick algorithm can be used to efficiently search for multiple patterns in a large blob of text, making it a really useful algorithm in data science and many other areas. 
This algorithm is helpful to find all occurrences of all given sets of keywords. It is a kind of Dictionary-matching algorithm. It uses a tree structure using all keywords. After making the tree, it tries to convert the tree as an automaton to do the searching in linear time. Our web app is required to search for PAM sequence patterns in a large string of target DNA in an efficient manner hence we decided to use this algorithm for the same.

Backend

Why Django for the backend?

Django is a free and open-source framework that is compatible with major operating systems and databases. It has a great number of advantages that made it one of the most popular technologies used for software development. It is considered exceptionally user-friendly, secure, clean, and efficient.

Ease of use

Django is considered a developer-friendly framework easy to pick up, especially for those developers who are comfortable with Python’s syntax.

Security

Django provides robust security features. It helps to avoid the common mistakes of web development including SQL injection, clickjacking, cross-site request forgery and scripting.

Clean And Efficient

Django helps write clean and efficient code. This framework is the best solution to create a minimum viable product (MVP) because it includes all of the necessary tools for creating any additional features for the product.

Future Directions

  • 1. Provide access to DNA sequences using their Gene ID as mentioned on NCBI website
  • 2. Improve the user experience of the web-tool

References:

  1. Prykhozhij, S., Rajan, V., Gaston, D. and Berman, J., 2022. CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences
  2. Collias, D., Beisel, C.L. CRISPR technologies and the search for the PAM-free nuclease. Nat Commun 12, 555 (2021).
    https://doi.org/10.1038/s41467-020-20633-y
  3. https://github.com/roshan-eng/iGEM_WebApp.git