High-Specificity Cas9 Nuclease Variant with tRNA-Processed Guide RNA
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Solution Overview
Problem
Current CRISPR/Cas9 genome editing systems suffer from low specificity and off-target effects, limiting their application due to inefficient site-directed modification of target sequences in organisms.
Innovation Solution
A genome editing system utilizing a high-specificity Cas9 nuclease variant fused with a tRNA or ribozyme guide RNA fusion, where the guide RNA is precisely processed to remove extra nucleotides at the 5' end, enhancing editing efficiency and specificity by improving transcription initiation and expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type Cas9 nuclease is used for genome editing, then editing efficiency is high, but specificity is low and off-target effects occur
Solution Approach 1:
The patent applies parameter changes by modifying the Cas9 nuclease through specific amino acid substitutions (e.g., K810A, K1003A, R1060A in eSpCas9; N497A, R661A, Q695A, Q926A in SpCas9-HF1) to alter its binding and cleavage properties. These parameter changes in the Cas9 protein structure enable high-specificity variants to maintain editing efficiency while dramatically improving specificity and reducing off-target effects compared to wild-type Cas9.
2Reliability
If high-specificity Cas9 nuclease variant is used, then specificity is improved, but editing efficiency decreases
Solution Approach 1:
The patent introduces tRNA-guide RNA fusion as an intermediary mechanism. The tRNA component serves as a mediator that is precisely processed by cellular enzymes (RNase P and RNase Z) to generate mature guide RNA with exact 5' and 3' ends. This intermediary tRNA structure ensures complete complementarity between guide RNA and target DNA, enabling high-specificity Cas9 variants to achieve editing efficiency comparable to wild-type Cas9 while maintaining their superior specificity.
Solution Approach 2:
The patent applies preliminary action by pre-fusing the guide RNA to tRNA in a structured format before cellular processing. This preliminary fusion ensures that the guide RNA sequence is already precisely defined and protected, allowing cellular processing enzymes to generate perfectly matched guide RNA molecules that maximize binding specificity and editing efficiency when used with high-specificity Cas9 variants.
3Ease of manufacture
If extra nucleotides are present at 5' end of guide RNA, then transcription initiation is simplified, but binding specificity to target sequence decreases
Solution Approach 1:
The tRNA acts as an intermediary that solves the contradiction between simplified transcription initiation and precise binding. The tRNA fusion allows standard RNA polymerase III transcription to proceed without special considerations for 5' end nucleotides, while the tRNA processing enzymes (RNase P and RNase Z) precisely cleave to remove any extra nucleotides, ensuring the mature guide RNA has exact complementarity to the target sequence.
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 achieves editing efficiencies comparable to wild-type Cas9 while maintaining high specificity, reducing off-target effects and expanding the range of targetable sequences, thereby improving the precision and effectiveness of genome editing.
Implementation Method 1
Cas9 protein cleaves a specific DNA sequence under the guidance of a gRNA to create a double-strand break (DSB)
Implementation Method 2
the fusion is cleaved at the 5′ end of the guide RNA after being transcribed in the cell, thereby forming a guide RNA that does not carry extra nucleotide at the 5′ end
Implementation Method 3
the first ribozyme is designed to cleave the fusion at the 5′ end of the guide RNA, thereby forming a guide RNA that does not carry extra nucleotide at the 5′ end
Implementation Method 4
a gRNA that is complementary to a target sequence
Data Source
AI summary
The present invention relates to the field of genetic engineering. In particular, the present invention relates to a genome editing method with high efficiency and high specificity. More specifically, the present invention relates to a method for increasing the efficiency of site-directed modification of a target sequence in a genome of an organism by a high-specificity Cas9 nuclease variant.