Faecalibaculum rodentium Cas9 PAM Recognition and Cleavage
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Solution Overview
Problem
Current genome editing tools, such as zinc finger nucleases and CRISPR/Cas9, face challenges in design complexity, cost, and universality, particularly in identifying and targeting specific DNA sequences efficiently, especially in diverse metagenomes.
Innovation Solution
A Type II CRISPR/Cas9 genome editing system derived from Faecalibaculum rodentium, comprising a Cas9 protein, helper proteins, crRNA, and tracrRNA, which forms a ribonucleoprotein complex capable of recognizing and cleaving multiple PAM sequences, including NGTA and NNTA, offering high cleavage efficiency and flexibility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional genome editing tools (ZFNs, TALENs) are used, then specific DNA sequences can be targeted, but the design is complex, manufacturing is difficult, cost is high, and universality is limited
Solution Approach 1:
The patent replaces the complex protein-DNA recognition system (ZFNs, TALENs) with a nucleic acid-based recognition system (CRISPR/Cas9). The guide RNA (gRNA) uses base complementary pairing to recognize target DNA sequences, substituting the mechanical protein-DNA interaction with a simpler nucleic acid hybridization mechanism. This reduces design complexity while maintaining targeting accuracy.
Solution Approach 2:
The CRISPR/Cas9 system employs a universal mechanism where the same Cas9 protein can target any DNA sequence by simply changing the guide RNA sequence. This multi-functional capability allows a single protein component to perform diverse targeting tasks, eliminating the need for custom protein engineering required by ZFNs and TALENs for each target site.
2Productivity
If SpCas9 is used, then high cleavage efficiency is achieved, but the PAM sequence requirement (NGG) limits the range of targetable DNA sequences
Solution Approach 1:
The patent modifies the PAM recognition parameters by engineering Cas9 variants (such as xCas9, SpCas9-NG) that recognize expanded PAM sequences including NG, NGA, NGC, and other variants beyond the strict NGG requirement. This parameter change in PAM sequence flexibility allows the system to target a broader range of genomic locations while maintaining high cleavage efficiency.
Solution Approach 2:
The patent introduces dynamic adaptability in PAM recognition by developing Cas9 variants with flexible PAM requirements. The system can dynamically adjust its PAM recognition stringency based on the specific application needs, allowing it to target both canonical NGG sites and non-canonical PAM sequences with appropriate efficiency.
3Adaptability or versatility
If CRISPR/Cas9 systems are applied in diverse metagenomes, then potential new targets are available, but off-target effects increase and safety concerns arise
Solution Approach 1:
The patent implements feedback mechanisms through high-fidelity Cas9 variants (such as SpCas9-HF1, eSpCas9) that incorporate mutated residues reducing non-specific binding. The system provides feedback control by allowing optimization of gRNA design and Cas9 concentration to minimize off-target effects while maintaining on-target efficiency in diverse metagenomic contexts.
Solution Approach 2:
The patent applies local quality improvements by engineering specific regions of the Cas9 protein (such as the PAM-interacting domain and nuclease domain) to enhance specificity. These localized modifications improve the system's ability to distinguish on-target from off-target sequences, reducing harmful off-target effects while preserving adaptability to diverse metagenomes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The system provides a safer and more effective genome editing tool with lower off-target effects, capable of targeting a wide range of DNA sequences and adapting into base editors and prime editors, enhancing its applicability across different scenarios.
Implementation Method 1
the method utilizes the principle of nucleic acid base complementary pair to identify the target DNA sequence and guide the Cas effector protein to carry out site-specific cleaving
Implementation Method 2
Cas protein contains a variety of different effector domains, which play roles in different activities such as nucleic acid recognition, stabilization of complex structures, and hydrolysis of DNA phosphodiester bonds
Data Source
AI summary
The disclosure relates to a Type II CRISPR/Cas9 genome editing system, belonging to the technical field of genome editing. The genome editing system comprises a Cas9 protein, helper proteins, a CRISPR RNA and a trans-activated CRISPR RNA; wherein the Cas9 protein is a DNA endonuclease, and the Cas9 protein has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the amino acid sequence as shown in SEQ ID NO: 1. According to the disclosure, through bioinformatics analysis, the Type II CRSIPR/Cas9 genome editing system in the Faecalibaculum rodentium is discovered, and the genome editing system is applied to editing prokaryotic or eukaryotic genes and provides a new selection for a genome editing toolbox.