Guide RNA Scaffold with Primer Binding Site for In Situ Sequencing

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Solution Overview

Problem

Current methods for identifying guide RNA spacer sequences within cells are limited and lack efficient techniques for sequencing and amplification, hindering the precise modulation of CRISPR-Cas systems for gene therapy and genomic modifications.

Innovation Solution

Incorporating a reverse transcription primer binding site within the scaffold sequence of guide RNA, allowing for in situ sequencing and amplification using methods like FISSEQ, which enables the identification and sequencing of spacer sequences while maintaining the guide RNA's functionality.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If guide RNA spacer sequences are identified using conventional methods, then the identification process is simple, but the precision and comprehensiveness of sequencing is insufficient

Engineering Contradiction:

Improvesequencing precisionVSAvoidmethod complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent introduces an intermediary reverse transcription primer binding site sequence within the scaffold sequence that mediates between the guide RNA spacer sequence and the sequencing methodology. This intermediary element enables FISSEQ and other sequencing methods to accurately identify and sequence spacer sequences by providing a specific binding target for reverse transcription primers, thereby resolving the contradiction between sequencing precision and method complexity.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent applies preliminary action by pre-incorporating the reverse transcription primer binding site sequence into the scaffold sequence during guide RNA design. This preliminary preparation enables subsequent in situ sequencing and amplification procedures to proceed efficiently with high precision, avoiding the need for complex post-processing steps and reducing overall methodological complexity.

Inventive Principle:

Principle #10Preliminary action

2Adaptability or versatility

If the scaffold sequence is modified to enable sequencing, then the guide RNA's functionality is maintained, but the structural complexity of the guide RNA increases

Engineering Contradiction:

Improveguide RNA functionalityVSAvoidguide RNA structure

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent applies universality by designing the scaffold sequence to serve multiple functions: maintaining its original role in guide RNA structure and function while simultaneously incorporating the reverse transcription primer binding site sequence to enable sequencing and amplification. This multi-functionality allows the same scaffold sequence to support both CRISPR-Cas system operation and molecular biology procedures without requiring separate components.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent merges the sequencing-enabled reverse transcription primer binding site sequence with the functional scaffold sequence into a single integrated structure. This combining approach allows the guide RNA to possess both sequencing capability and biological function within one molecule, reducing the need for additional separate components and simplifying the overall system architecture.

Inventive Principle:

Principle #5Merging (Combining)

3Measurement precision

If in situ sequencing methods are used to identify spacer sequences, then the precision of identification is improved, but the time and resources required increase

Engineering Contradiction:

Improveidentification precisionVSAvoidsequencing time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

Inventive Principle:

Principle #10Preliminary action

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

Enables precise identification and sequencing of guide RNA spacer sequences, facilitating the modulation of CRISPR-Cas systems for targeted gene modifications and enhancing the versatility of CRISPR technology.

Implementation Method 1

the reverse transcription primer binding site sequence or docking site sequence is at a location within the scaffold sequence which facilitates reverse transcription of the spacer sequence into a cDNA sequence

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

The spacer sequence hybridizes to the protospacer sequence of the target nucleic acid

Methodology Applied

Scientific EffectHybridization:

Implementation Method 3

The sequence of a gRNA region, called the spacer, determines the identity of the target locus

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS11732258B2Engineered guide RNA sequences for in situ detection and sequencing

Publication Date: 2023.08.22 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

A functional engineered guide RNA sequence is provided including a spacer sequence and a scaffold sequence, wherein the scaffold sequence includes a primer binding site for reverse transcription.