CRISPR-Cas Transposon Gene Targeting Without Double-Strand Breaks
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing CRISPR-Cas systems for chromosome editing are inefficient and prone to off-target events due to a cumbersome two-step process involving DNA breakage and recombination, which can be improved for more precise and efficient gene targeting.
Innovation Solution
A modified TnsA protein, adapted from Aeromonas salmonicida strain S44, is used in combination with Cas8/5f, Cas7f, and Cas6f to facilitate guide RNA-directed transposition of DNA cargo into chromosomes or extrachromosomal elements without inducing double-strand breaks, utilizing Tn7-like transposons and CRISPR-Cas systems for precise insertion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a two-step CRISPR-Cas process involving DNA breakage and recombination is used, then gene targeting can be achieved, but the process becomes cumbersome and inefficient with off-target events
Solution Approach 1:
The patent combines the CRISPR-Cas system with a transposon system into a single integrated platform. The Cas proteins (Cas8f, Cas5f, Cas7f, Cas6f) work together with transposon components (TnsA, TnsB, TnsC, TniQ) to achieve both targeted localization and DNA insertion in one coordinated process, eliminating the need for separate breakage and recombination steps.
Solution Approach 2:
The transposon system acts as an intermediary mechanism that bridges the gap between CRISPR-Cas target recognition and stable DNA integration. Instead of relying on host recombination systems to repair breaks, the transposon provides a dedicated insertion pathway that is activated by CRISPR-Cas guided cleavage, streamlining the overall process.
2Manufacturing precision
If CRISPR-Cas systems are used to break host chromosomes for DNA insertion, then gene editing can occur, but off-target events increase and precision decreases
Solution Approach 1:
The system uses the CRISPR-Cas complex itself to generate the insertion site rather than relying on separate endonuclease activities. The Cas proteins guided by specific RNA sequences create nicks or breaks precisely at the intended target location, and the transposon immediately utilizes this site for insertion, ensuring that the breaking and insertion occur at the same precise location without generating off-target breaks.
Solution Approach 2:
The CRISPR-Cas complex performs preliminary target recognition and binding to the specific DNA sequence before the actual insertion occurs. This preliminary action ensures that the transposon is directed to the correct location, and the subsequent breakage and insertion are confined to this pre-determined site, preventing off-target events.
3Productivity
If a modified TnsA protein is used with Cas proteins for transposition, then transposition efficiency increases, but the system requires multiple protein components
Solution Approach 1:
The modified TnsA protein serves multiple functions within the system: it acts as a transposase for DNA movement, interacts with the CRISPR-Cas complex for target recognition, and facilitates the integration of the transposon at the guided location. This multi-functionality reduces the need for separate specialized proteins for each step of the process.
Solution Approach 2:
The system creates a composite functional complex by combining the modified TnsA transposon protein with the Cas8f, Cas5f, Cas7f, and Cas6f proteins. This composite assembly integrates the catalytic activities of transposition with the targeting capabilities of CRISPR-Cas, achieving high efficiency while consolidating multiple functions into a coordinated protein complex.
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 method achieves increased transposition efficiency and precision, allowing for targeted DNA insertion up to 49 nucleotides away from the protospacer, demonstrating functionality in both prokaryotic and eukaryotic cells, including E. coli and eukaryotic organisms.
Implementation Method 1
a transposable DNA cargo sequence... and at least one guide RNA comprising a spacer targeted to a target DNA sequence in the chromosome or the extrachromosomal element. The DNA target sequence comprises a protospacer and a protospacer adjacent motif (PAM) that is 5′ to the protospacer. Thus, the spacer in the guide RNA may be complementary to a protospacer.
Implementation Method 2
The disclosure includes use of a modified TnsA protein for which an unmodified counterpart was initially identified as encoded by Aeromonas salmonicida strain S44, on its plasmid pS44-1... The modified TnsA protein functions in the context of transposon proteins TnsB, TnsC, TniQ, and Cas proteins Cas8f, Cas5f, Cas7f, Cas6f... facilitates the guide-RNA directed transposition of any desired DNA cargo into the chromosome or extrachromosomal element
Data Source
AI summary
Provided are compositions and methods for transposon-CRISPR-based DNA editing. The compositions and methods include a modified TnsA protein that is altered from its original sequence found in Aeromonas salmonicida, and provides improved transposition frequency, and is functional in a heterologous system. The TnsA protein is used in a system with transposon proteins TnsB, TnsC, TniQ, Cas proteins Cas8f, CasSf, Cas7f, Cas6f, a transposable DNA cargo sequence that is flanked by left and right transposon sequences, and at least one guide RNA that contains at least one spacer targeted to a target DNA sequence in the chromosome or the extrachromosomal element.