Engineered Cas9 PAM Specificity for Broader Genome Targeting
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Solution Overview
Problem
The existing CRISPR-Cas9 nucleases, such as Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9), have limited targeting range due to specific protospacer adjacent motif (PAM) recognition, constraining efficient genome editing in various organisms and cell types.
Innovation Solution
Engineered variants of SpCas9 and SaCas9 with altered PAM specificities are developed through structural information, bacterial selection-based directed evolution, and combinatorial design, enabling robust editing of endogenous gene sites in zebrafish and human cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Cas9 uses a specific PAM recognition mechanism, then sequence-specific cleavage is achieved, but the targeting range is constrained
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (D1135, G1218, T1337) in the PAM-interacting domain of Cas9 to alter its PAM specificity. These mutations change the biochemical parameters of the protein to enable recognition of novel PAM sequences while maintaining sequence-specific cleavage precision through the guide RNA mechanism.
Solution Approach 2:
The patent introduces dynamics by creating a flexible PAM recognition system where the Cas9 protein can adapt to different PAM sequences through engineered mutations. The system transitions from a static, fixed PAM recognition mechanism to a dynamic one that can be reconfigured through protein engineering to target different genomic locations.
2Measurement precision
If Cas9 recognizes longer PAM sequences, then targeting specificity is improved, but the number of targetable sites decreases
Solution Approach 1:
The patent uses parameter changes to optimize the balance between PAM length and targetable sites. By mutating specific residues in the PAM-interacting domain, the system can adjust its PAM recognition parameters to achieve optimal specificity while maintaining sufficient target availability in the genome.
Solution Approach 2:
The patent applies local quality by making targeted changes only in the specific amino acid residues responsible for PAM recognition (D1135, G1218, T1337), while leaving the rest of the Cas9 protein structure unchanged. This localized modification allows optimization of PAM specificity without affecting the overall functionality and targetable site quantity.
3Reliability
If wild-type SpCas9 is used, then canonical NGG PAM sites are targeted, but off-target effects occur at non-canonical PAM sites
Solution Approach 1:
The patent converts the potential harm of non-specific PAM recognition into a benefit by engineering Cas9 variants that selectively recognize specific non-canonical PAM sequences. The mutations in the PAM-interacting domain transform the protein's recognition properties to distinguish between canonical and non-canonical PAMs, converting what was previously harmful non-specific binding into specific, controlled targeting.
Solution Approach 2:
The patent implements feedback through directed evolution and structural information feedback loops. The PAM-interacting domain is engineered based on structural data and selection pressure from experimental systems, allowing iterative optimization of PAM specificity to reduce off-target effects while maintaining on-target reliability.
Data Source
Figure 1A~1D
Figure 1E~1F
Figure 1G~1I
AI summary
Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.