Compact ENQP Cas Proteins for Single-AAV Genome Editing
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Solution Overview
Problem
The challenge of packaging large Cas proteins like SpCas9 with guide RNAs into a single AAV vector is hindered by the limited packaging capacity of AAVs, and there is a need for smaller Type II Cas nucleases with diverse PAM specificities to enhance genome editing flexibility and efficiency.
Innovation Solution
Development of Type II Cas proteins, such as ENQP Type II Cas proteins, which are significantly shorter than SpCas9, and can be packaged with gRNAs in a single AAV vector, along with fusion proteins and chimeric designs to broaden targetable sites in the cell genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If large Cas proteins like SpCas9 are packaged with guide RNAs into a single AAV vector, then genome editing capability is achieved, but packaging capacity is exceeded
Solution Approach 1:
The patent extracts the core functional domains from the large SpCas9 protein to create a minimized Cas9 variant. By removing non-essential regions and retaining only the critical domains (REC, HNH, RuvC, and PAM-interacting domains), the protein size is reduced to fit within AAV packaging capacity while preserving genome editing functionality
Solution Approach 2:
The patent changes the physical parameter of protein size by creating truncated and minimized versions of Cas9. The amino acid sequence is modified to reduce length from the original SpCas9 size to a compact form that can be packaged with gRNA in a single AAV vector, while maintaining catalytic activity through preservation of key domains
2Adaptability or versatility
If Type II Cas nucleases with diverse PAM specificities are developed, then targetable sites in the genome are increased, but protein size and packaging complexity increase
Solution Approach 1:
The minimized Cas9 protein is designed to recognize multiple PAM sequences (NGG, NGAG, NGCG) through engineered variations in the PAM-interacting domain. This multi-functional capability allows a single compact protein to target diverse genomic sites with different PAM specificities, eliminating the need for multiple specialized nucleases
Solution Approach 2:
The Cas9 protein is segmented into functional domains (REC for recognition, HNH and RuvC for cleavage, PAM-interacting domain for targeting). By optimizing and minimizing each segment independently, the patent achieves a compact overall structure that can accommodate diverse PAM recognition capabilities without increasing total protein size
Data Source
AI summary
Type II Cas proteins referred to as ENQP Type II Cas proteins; gRNAs for Type II Cas proteins; systems comprising Type II Cas proteins and gRNAs; nucleic acids encoding the Type II Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.