CRISPR-Cas Molecular Recording System for Arbitrary DNA Storage

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Solution Overview

Problem

Current systems for recording information in living cells are limited in their ability to encode and store arbitrary DNA sequences, with most approaches only capable of storing little more than a single byte of information, and there is a need for a CRISPR-Cas system that can efficiently direct the recording of specific and arbitrary DNA sequences into both prokaryotic and eukaryotic cells.

Innovation Solution

The introduction of a method that uses the CRISPR-Cas system by providing cells with nucleic acid sequences encoding Cas1 and Cas2 proteins, along with a CRISPR array and oligonucleotide sequences, allowing for the specific and arbitrary recording of DNA sequences into the genome, utilizing the Cas1-Cas2 complex to integrate synthetic oligo spacers into the genome, enabling multi-modal molecular recording.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If recombinases are used to store information via orientation of DNA segments, then information can be stored permanently in the genome, but the recording capacity is limited to binary states (little more than a single byte)

Engineering Contradiction:

Improvepermanent storageVSAvoidrecording capacity

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The invention segments the DNA recording system into distinct functional components: Cas1-Cas2 complex for spacer acquisition, CRISPR array for storage, and guide RNA for retrieval. This segmentation allows each component to be optimized independently, enabling the system to store arbitrary DNA sequences rather than being limited to binary states, thereby increasing recording capacity while maintaining permanent storage capability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The CRISPR-Cas system is designed to perform multiple functions: it can acquire spacers from any exogenous DNA source (universal spacer acquisition), store them in the CRISPR array (memory function), and use them for targeted DNA cleavage (immune function). This multi-functionality allows the system to record arbitrary DNA sequences of any length, overcoming the binary limitation of recombinase-based systems

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If transcriptional level encoding with feedback loops is used, then cellular memory can be created, but the information storage capacity remains limited

Engineering Contradiction:

Improvememory functionVSAvoidinformation capacity

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The invention transitions from transcriptional level encoding (one-dimensional, binary on/off states) to direct DNA sequence encoding (multi-dimensional, utilizing all four nucleotide bases). By embedding arbitrary DNA sequences directly into the genome at specific loci, the system achieves vastly increased information capacity while maintaining the memory function through stable genomic integration

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

3Adaptability or versatility

If arbitrary DNA sequences are recorded into the genome, then information capacity increases significantly, but the complexity of the recording system increases

Engineering Contradiction:

Improveencoding capacityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The Cas1-Cas2 complex autonomously acquires spacers from exogenous DNA and integrates them into the CRISPR array without requiring external intervention. The system self-regulates spacer acquisition, processing, and integration, reducing the need for complex external recording machinery while enabling high-capacity arbitrary DNA sequence storage

Inventive Principle:

Principle #25Self-service

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

This approach allows for the recording of specific and arbitrary DNA sequences with a capacity exceeding previous synthetic biological memory systems, enabling the storage of over 100 bytes of information in bacterial genomes and providing a robust method for molecular recording and decoding.

Implementation Method 1

providing the cell with a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system, providing the cell with a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the cell expresses the Cas1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell

Methodology Applied

Scientific EffectCRISPR-Cas system:

Data Source

PatentUS11326161B2Methods and systems of molecular recording by CRISPR-Cas system

Publication Date: 2022.05.10 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

This invention provides methods of altering a cell including providing the cell with a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system, providing the cell with a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the cell expresses the Cas1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid. Also provided are methods and systems for nucleic acid storage and in vivo molecular recordings of events into a cell.