Circularly Permuted Cas9 Variants for Expanded PAM Recognition
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Solution Overview
Problem
Current CRISPR-Cas9 genome editing technologies are limited by strict protospacer-adjacent motif (PAM) requirements and a narrow editing window, restricting the sequences that can be targeted and edited, particularly for adenine base editors, which hampers the scope of genome and base editing applications.
Innovation Solution
Development of circularly permuted Cas9 variants with expanded PAM recognition capabilities and increased editing windows, allowing for non-canonical PAM sequence recognition and broader editing windows within and outside the protospacer sequence, enhancing the scope of genome and base editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If canonical Cas9 is used for base editing, then the PAM recognition is specific (5'-NGG-3'), but the targeting scope is limited
Solution Approach 1:
The patent applies parameter changes by modifying the PAM recognition interface of Cas9 through circular permutation. Specifically, the Cas9 protein is circularly permuted at different positions (e.g., CP1012, CP1028, CP1041, CP1249, CP1300) to alter how it recognizes PAM sequences. This structural reconfiguration allows the enzyme to recognize non-canonical PAM sequences (such as 5'-NGA-3', 5'-NGT-3') while maintaining sufficient specificity for reliable genome editing applications.
2Adaptability or versatility
If standard Cas9 base editors are used, then the editing window is narrow (4-5 bases), but the targeting scope is restricted
Solution Approach 1:
The patent extends the editing window parameter by circularly permuting Cas9 at positions that relocate the catalytic domain relative to the PAM recognition domain. This structural adjustment allows the editing window to expand from the canonical 4-5 bases to include positions up to 8-9 bases away from the PAM site, thereby increasing the number of targetable positions while maintaining editing precision through the preserved catalytic mechanism.
3Adaptability or versatility
If circularly permuted Cas9 variants are used, then the editing window expands to 8-9 bases, but the protein structure is modified
Solution Approach 1:
The patent applies segmentation by dividing the Cas9 protein into functional domains and reconfiguring them through circular permutation. The Cas9 sequence is cleaved at specific positions (e.g., between residues 1011-1012 for CP1012) and reconnected to form a circularly permuted structure. This segmentation allows the PAM recognition domain and catalytic domain to be spatially rearranged, expanding the editing window while preserving the essential functional interactions through carefully selected permutation sites.
4Adaptability or versatility
If non-canonical PAM sequences are recognized, then the targeting scope increases, but the editing efficiency may be reduced
Solution Approach 1:
The patent optimizes the balance between targeting scope and editing efficiency by selecting specific circular permutation positions that minimize structural disruptions to the catalytic mechanism. The CP variants (e.g., CP1012, CP1028, CP1041, CP1249, CP1300) are designed to recognize non-canonical PAM sequences while maintaining proper positioning of the catalytic residues, thereby preserving base editing efficiency even when targeting sequences with relaxed PAM requirements.
Data Source
AI summary
The present disclosure provides improved adenosine base editors (ABE) that have an expanded range of PAM sequence recognition capability (i.e., recognition of non-canonical ′5-NGG-′3 PAM sequence). In addition, the present disclosure provides improved cytidine base editors (CBE) and adenosine base editors (ABE) comprising circular permutant variants of Cas9 (CP-Cas9) with an increased window of base editing within the protospacer sequence (e.g., from about 4-5 nucleotides to up to about 8-9 nucleotides) and even outside of the protospacer sequence.