Engineered ScCas9 Variants for Single-Base PAM Targeting
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Solution Overview
Problem
The limitations of existing CRISPR-Cas9 systems, such as Streptococcus pyogenes Cas9 (SpCas9), in targeting specific genomic sequences due to stringent protospacer adjacent motif (PAM) requirements, constrain the applicability of genome editing applications like base editing and homology-directed repair, necessitating the development of variants with broader PAM specificities.
Innovation Solution
Engineering Streptococcus canis Cas9 (ScCas9) variants, specifically Sc+ and Sc++, by incorporating amino acid mutations and a novel DNA-interacting loop domain, to enhance PAM flexibility and targeting efficiency, allowing for 5′-NG-3′ and 5′-NNG-3′ genomic sequence recognition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Streptococcus pyogenes Cas9 (SpCas9) is used for genome editing, then high editing efficiency is achieved, but the targeting range is limited by stringent PAM requirements (5′-NGG-3′)
Solution Approach 1:
The patent applies parameter changes by modifying the PAM recognition requirements of Cas9 through protein engineering. Specifically, it introduces variants with relaxed PAM specificities (5′-NG-3′, 5′-NNG-3′, 5′-NRG-3′) by altering amino acid residues in the PAM-interacting domain, thereby expanding the targeting range while maintaining editing efficiency
Solution Approach 2:
The patent achieves universality by creating a family of Cas9 variants that can recognize multiple different PAM sequences. The engineered ScCas9 variants can target diverse genomic locations with different PAM motifs, making the system universally applicable across various genome editing applications including base editing and homology-directed repair
2Adaptability or versatility
If Cas9 variants with relaxed PAM specificity are engineered, then broader targeting range is achieved, but fidelity may be reduced
Solution Approach 1:
The patent applies local quality by making specific localized modifications to the PAM-interacting domain of Cas9 while preserving the rest of the protein structure. This allows the variant to recognize relaxed PAM sequences at the binding interface while maintaining high fidelity cutting at the target site through the unchanged catalytic and guide RNA binding regions
3Adaptability or versatility
If engineered ScCas9 variants (Sc+, Sc++) are used, then broader PAM recognition (5′-NG-3′, 5′-NNG-3′) is achieved, but protein structure complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the Cas9 protein into functional domains, specifically focusing modifications on the PAM-interacting domain while leaving other domains unchanged. This modular approach allows independent optimization of PAM recognition without disrupting the overall protein structure and function
Data Source
AI summary
Engineered Streptococcus canis Cas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition of Streptococcus pyogenes Cas9 (SpCas9-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE [SEQ ID No. 4] in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5′-NG-3′ or 5′-NNG-3′ and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.