Cas9 Polypeptide Variants for Expanded PAM Recognition

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Solution Overview

Problem

Current synthetic type II CRISPR-Cas systems face challenges in efficiency and specificity for site-specific cleavage, nicking, transcriptional control, and genome editing due to limitations in target recognition and cleavage mechanisms.

Innovation Solution

A protein-RNA complex comprising a Cas9 polypeptide with at least 80% identity to specific amino acid sequences and a CRISPR RNA with defined regions for sequence specificity, combined with a tracrRNA for enhanced target binding and cleavage, is used for site-specific cleavage, nicking, and transcriptional control, and editing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If current synthetic type II CRISPR-Cas systems are used for site-specific cleavage and genome editing, then the basic functionality is achieved, but the efficiency and specificity are insufficient

Engineering Contradiction:

ImprovespecificityVSAvoidefficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent modifies key parameters of the CRISPR-Cas system including the PAM sequence recognition (changing from strict NGG requirement to accepting NG, NGAA, NNG, NGA, NTAA, NTG, NNC, NNAAC sequences), altering the spacer length (13-30 nucleotides), and modifying Cas9 protein sequences (80-95% identity to Streptococcus thermophilus Cas9) to improve both efficiency and specificity of target recognition and cleavage

Inventive Principle:

Principle #35Parameter changes

2Reliability

If the CRISPR-Cas system is designed for high specificity through strict target recognition, then specificity is improved, but the complexity of the system increases

Engineering Contradiction:

ImprovespecificityVSAvoidsystem complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent creates Cas9 variants with relaxed PAM sequence requirements that can recognize multiple PAM types (NG, NGAA, NNG, NGA, NTAA, NTG, NNC, NNAAC), allowing a single Cas9 protein to perform multiple target recognition functions. The system also accommodates varied spacer lengths (13-30 nt) and can be used for multiple applications including cleavage, nicking, and transcriptional control, reducing the need for separate specialized components

Inventive Principle:

Principle #6Universality (Multi-functionality)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The solution significantly enhances the efficiency and specificity of site-specific cleavage, nicking, and transcriptional control, allowing precise editing of target DNA sequences by improving the recognition and binding capabilities of the CRISPR-Cas system.

Implementation Method 1

the spacer sequence hybridizes with a portion of a target DNA that is complementary to the spacer sequence

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

at least a portion of the 5' region of the tracrRNA is complementary to the 3' region (CRISPR repeat) of the crRNA

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 3

Cas endonucleases (Garneau et al. 2010. Nature. 468:67-71; Haurwitz et al. 2010. Science. 329:1355-1358)

Methodology Applied

Scientific EffectNuclease cleavage: Enzyme

Data Source

PatentUS11753651B2Cas9 proteins and guiding features for DNA targeting and genome editing

Publication Date: 2023.09.12 NORTH CAROLINA STATE UNIV

AI summary

The present invention is directed to methods and compositions comprising novel CRISPR polypeptides and polynucleotides for site-specific cleavage and nicking of nucleic acids, transcriptional control and genome editing.