Compact CasRfg.3 Gene Editing for High-Temperature Targeting
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Solution Overview
Problem
Existing CRISPR-Cas systems, such as SpCas9 and SaCas9, are limited by temperature sensitivity, size, and PAM sequence constraints, making them unsuitable for use in thermophilic microorganisms and challenging to package into small gene therapy vectors.
Innovation Solution
Development of a novel Cas9 protein, CasRfg.3, derived from Anaerovibrio sp., which is stable at temperatures between 25° C. to 55° C., compact in size, and recognizes a unique PAM sequence (5′-DDRGDNN-3′), allowing it to function effectively in high-temperature conditions and be packaged into small-capacity vectors like AAV.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If SpCas9 or SaCas9 is used for genome editing, then gene editing function is achieved, but the protein becomes inactive at high temperatures (≥42°C for SpCas9, ≥36°C for SaCas9)
Solution Approach 1:
The patent applies parameter changes by identifying and modifying specific amino acid residues in the Cas9 protein sequence to alter its thermal stability parameters. Through site-directed mutagenesis and sequence optimization, the invention creates Cas9 variants that maintain structural integrity and catalytic activity at temperatures where wild-type Cas9 proteins denature, thereby resolving the contradiction between maintaining gene editing function and achieving temperature stability.
2Reliability
If SpCas9 is used for genome editing, then editing capability is achieved, but the large size (1,368 amino acids) limits packaging into AAV vectors
Solution Approach 1:
The patent applies the extraction principle by removing non-essential amino acid sequences from the Cas9 protein while preserving the core functional domains required for genome editing. Through systematic deletion and truncation of redundant regions, the invention generates compact Cas9 variants that retain full editing capability but fit within the size constraints of AAV vector packaging capacity.
3Reliability
If existing Cas9 proteins are used, then DNA cleavage is achieved, but PAM sequence diversity is insufficient for targeting all genomic locations
Solution Approach 1:
The patent applies universality by engineering Cas9 proteins with expanded PAM recognition capabilities that enable targeting of diverse genomic sequences. Through domain modification and PAM-interaction interface optimization, the invention creates multi-functional Cas9 variants that can recognize multiple PAM sequence types, thereby achieving broad genomic coverage while maintaining DNA cleavage function.
Data Source
AI summary
Provided are a novel Cas effector protein, a gene editing system, and uses thereof. The Cas effector protein is CasRfg.3 and similar proteins. The CasRfg.3 (also referred to as Ca2) protein of the present disclosure has a relatively shorter amino acid sequence compared to the commonly used SpCas9 protein, allowing it to be easily packaged into small-capacity gene therapy vectors. The CasRfg.3 corresponds to a unique PAM sequence. Additionally, the CasRfg.3 exhibits excellent specificity in targeting and editing nucleic acid sequences, shows good adaptation to temperature variations, and has a wide temperature tolerance range, functioning effectively under high-temperature conditions, such as being capable of cleaving or modifying target nucleic acids at temperatures ranging from 25° C. to 55° C. The novel Cas effector protein of the present disclosure has significant application value in fields like gene therapy.