Cas9 Surface Loop Mutations for Extended sgRNA Specificity
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Solution Overview
Problem
Current CRISPR/Cas9 nucleases, particularly SpCas9 variants with increased fidelity, face limitations in target specificity and compatibility with extended guide RNAs, restricting their application due to reduced activity with 5' mismatched or extended sgRNAs, and limited target space, which hampers genome editing precision and efficiency.
Innovation Solution
Modifying a surface loop in the Cas9 protein to accommodate longer spacer sequences by disrupting the association between the 5' end of the spacer sequence and amino acids, allowing for increased target space and fidelity without impairing activity, enabling the use of 21-nucleotide long spacer sequences and improved specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If increased fidelity mutant variants of SpCas9 are used, then target specificity is improved, but target space is limited and compatibility with extended sgRNAs is reduced
Solution Approach 1:
The patent applies parameter changes by modifying the physical-chemical properties of the Cas9 protein through specific amino acid substitutions in the surface loop region. These mutations alter the protein's interaction interface with the sgRNA, changing its binding characteristics to accommodate extended spacer sequences while preserving off-target discrimination capability. This resolves the contradiction by tuning the protein's parameters to simultaneously achieve high specificity and extended target space compatibility.
Solution Approach 2:
The invention applies local quality by introducing mutations specifically in the surface loop region (residues 1004-1017) of the Cas9 protein, which is the local area responsible for interacting with the 5' end of the spacer sequence. By modifying only this specific local region rather than the entire protein, the invention maintains the global structural integrity and catalytic function of Cas9 while altering the local binding properties to accept extended sgRNAs, thus resolving the contradiction between specificity and adaptability.
2Adaptability or versatility
If 5'-extended sgRNAs are used with wild type SpCas9, then target space is expanded, but off-target activity increases
Solution Approach 1:
The patent applies preliminary action by pre-modifying the Cas9 protein structure through surface loop mutations before it encounters the target DNA. This preliminary structural adjustment creates a protein variant that is pre-adapted to recognize and bind extended sgRNA sequences with high fidelity. When this pre-modified Cas9 is subsequently used with 5'-extended sgRNAs, it maintains high target specificity without exhibiting increased off-target activity, thus resolving the contradiction between expanded target space and reduced off-target effects.
3Measurement precision
If increased fidelity mutant variants are used, then specificity is improved, but activity with extended sgRNAs is reduced
Solution Approach 1:
The invention applies universality by creating a Cas9 variant that performs multiple functions: it maintains the high specificity characteristic of increased fidelity mutants while simultaneously gaining the ability to effectively bind and process various sgRNA formats including 5'-extended sequences. The surface loop mutations confer multi-functionality on the Cas9 protein, enabling it to operate effectively across different sgRNA configurations without sacrificing activity, thus resolving the contradiction between specificity and productivity.
Data Source
AI summary
Some aspects of the present disclosure provide compositions, methods, and kits for improving the specificity and/or targetable sequence space of endonucleases programmable by RNA such as Cas9. Also provided herein are variants of Cas9 that have been engineered to have improved specificity for cleaving nucleic acid targets. Also provided herein are variants of Cas9 with increased fidelity that have been engineered to became compatible with 5′ extended sgRNAs such as 21G-sgRNAs. Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA (e.g., genomic modification), epigenomic engineering, transcriptome regulation, genome targeting.