In recent years, CRISPR gene editing technology has attracted great attention due to its enormous potential in the treatment of hereditary diseases. However, the potential off target effects that may occur during gene editing have always been an important safety concern. In addition, different populations have different genetic information, which poses great difficulties in accurately detecting off target effects. Today, the editor brings you a tool called CRISPRme that can be used for off target detection based on genetic variation information. This tool can consider genetic variation between individuals and more accurately predict and evaluate off target loci for gene editing.
1、 CRISPRme principle
The CRISPR Cas system is a natural bacterial immune mechanism used to resist foreign genetic materials such as bacteriophages or plasmids. In gene editing, Cas proteins (such as commonly used SpCas9) and gRNA are key components for achieving DNA cleavage. GRNA is a designed RNA molecule that contains a complementary spacer sequence to the target DNA sequence, allowing it to guide Cas proteins to specific genomic locations. CRISPRme simulates the DNA recognition and binding process of the CRISPR Cas system to predict potential off target sites.
Figure 1. CRISPR-CAS system
Supported by efficient computational models and algorithms, CRISPRme can handle a large amount of genetic variation data and genomic sequences. Firstly, CRISPRme utilizes the Aho Corasick automaton algorithm for multimodal string matching, allowing for simultaneous recognition of multiple PAM sequences, which is a key factor for Cas protein recognition and DNA binding in the CRISPR Cas system. Secondly, CRISPRme utilizes a Ternary Search Tree to establish a genome index, enabling it to quickly search and compare matches between genome sequences and gRNA interval sequences. Finally, CRISPRme performs a genome-wide scan to identify potential off target sites that match the gRNA spacer sequence, while evaluating the number and location of mismatches and RNA: DNA protrusions.
2、 Key steps and characteristics of CRISPRme off target detection
1. Integration of genetic variation: CRISPRme is capable of processing and integrating genetic variation information such as single nucleotide polymorphisms (SNPs) and insertion deletions (indices). These variant data typically come from databases such as the 1000 Genomes Project or the Human Genome Diversity Project (HGDP).
2. Reference genome and PAM sequence: Users can input specific Cas protein types, gRNA spacer sequences, PAM sequences, and genome construction information. CRISPRme uses this information to simulate the binding and cleavage of Cas protein and gRNA in the genome.
3. Search for off target sites: CRISPRme performs a genome-wide search to identify potential off target sites that match the gRNA interval sequence. It uses the Aho Corasick automaton algorithm to encode the PAM sequence and establishes an index using a ternary search tree to efficiently handle a large number of genetic mutations and off target searches.
4. Mismatch and bulge handling: CRISPRme allows users to define thresholds for mismatch and RNA: DNA bulges. These parameters will affect the accuracy and specificity of predicting off target sites.
5. Privacy and regulatory compliance: CRISPRme can be used online or deployed locally in protected and isolated environments, without transmitting or storing data online, thus respecting genetic privacy and regulations.
3、 CRISPRme operation
CRISPRme provides an easy-to-use online website（ http://crisprme.di.univr.it/ ）Researchers can directly log in to the website for off target detection and generate detailed reports, including mismatch and protrusion information for each potential off target site, as well as comparison results with user-defined genome annotations (such as GENCODE or ENCODE annotations). These reports can help researchers evaluate and prioritize off target sites with the highest risk.
In addition, the source code of CRISPRme has been uploaded to https://github.com/pinellolab/Crisprme , https://github.com/InfOmics/CRISPRme Users can use CRISPRme locally to perform off target detection and analysis of personalized genetic variation information.
CRISPRme, as a gene editing off target evaluation tool that considers genetic diversity, provides an important evaluation platform for future gene therapy. By combining genetic variation information from individuals and populations, CRISPRme can more accurately predict and evaluate the off target effects that gene editing may bring, thereby providing support for personalized healthcare and precision therapy.
CD Biosynsis is a leading gene editing service provider, providing services such as sgRNA design, amplicon sequencing, gene editing efficiency analysis, and off target detection. If you have any needs, please feel free to consult!
