Efficient and Seamless Plant Genome Insertion Technology is Officially Launched

On June 26, 2024, a collaborative team of R. Keith Slotkin from the University of Missouri and C. Nathan Hancock from the University of South Carolina published a research result titled Transposase-assisted target-site integration for efficient plant genome engineering in Nature. The study developed the transposase-assisted target site integration (TATSI) technology for efficient targeted insertion in plant genomes.

The researchers fused the rice Pong transposase protein with Cas9 or Cas12a nuclease, creating multiple different fusion protein configurations. Through gRNA guidance, these fusion proteins can achieve targeted insertion of the transposable element mPing. The experiment verified the insertion of mPing into the target site of the Arabidopsis PDS3 gene, demonstrating high insertion frequency and accuracy.

In addition, mPing can load different DNA elements (such as enhancers and gene cassettes) and successfully achieve targeted insertion of these elements, demonstrating the broad application prospects of this system in gene function research and crop improvement.

In Arabidopsis, the efficiency of targeted insertion using the one-component system was 35.5% (unfused ORF2 and Cas9 configuration).

In soybean, using the unfused ORF2 and Cas9 configuration, the insertion efficiency was 18.2%.

These insertion efficiencies are significantly higher than those of homology-directed repair (HDR) and non-homologous end joining (NHEJ) methods in plants.

In terms of insertion length: standard 430bp mPing element; 444bp mPing_HSE element containing six heat shock elements (HSEs); mPing_bar_CDS element containing the bar gene coding region (1002bp); mPing_bar element containing the bar gene expression cassette (including promoter and terminator, 1563bp); even tested an mPing_EPSPS element with a total length of 8.6kb, which contains two endogenous genes. The TATSI system is able to successfully insert these elements of different lengths.

Through deep sequencing analysis, the study found that most targeted insertion events occurred near the CRISPR cutting site (±4 bp), and the inserted mPing elements were mostly intact.

The study also evaluated the situation of off-target insertions and found that these off-target insertions were mainly due to free transposition events rather than non-specific cutting of CRISPR/Cas9.

First, let's understand how transposases work:

mPing transposon: mPing is a small, non-autonomous transposon with a length of 430bp and terminal inverted repeats (TIRs) at both ends. These TIRs are key sites for transposase recognition and binding.

Transposases ORF1 and ORF2: The movement of the mPing transposon requires two key proteins (ORF1 and ORF2). These two proteins are encoded by the Pong transposon and play an important role in the mPing transposition process. ORF1 is a Myb-like DNA binding protein that can recognize and bind to the TIR sequence of the mPing transposon. ORF2 is a transposase with a DDE catalytic motif that is responsible for the shearing and insertion of the mPing transposon.

ORF1 and ORF2 proteins recognize and bind to the TIR sequence of the mPing transposon to form a transposition complex. This complex ensures that the mPing transposon can be precisely cut and moved. After the mPing is cut, the ORF1 and ORF2 proteins continue to bind to the ends of the mPing, protecting them from degradation by nucleases. This ensures that the mPing remains intact when inserted into a new genomic location.

The one and two-component systems mentioned in the article:

Two-component system: contains donor elements, transposase and CRISPR/Cas system, where the donor element contains transposon mPing, which is pre-integrated into the plant genome. Transposase includes ORF1 and ORF2 proteins and Cas proteins, which are expressed by separate vectors. This system is mainly used in the verification stage.

One-component system: The one-component system integrates donor elements (mPing and exogenous DNA fragments), transposase ORF1 and ORF2, and CRISPR/Cas system into a single vector. This is mainly the state in practical application.

The structure of mPing when carrying different cargo:

mPing_HSE: Tests the ability of the TATSI system to carry enhancer sequences. HSEs are commonly used enhancer elements that can enhance gene expression.

mPing_bar_CDS: Tests the ability of the TATSI system to carry gene coding regions.

mPing_bar: Test the ability of the TATSI system to carry a complete gene expression cassette. The bar gene expression cassette can drive the expression of the bar gene in plants, making them resistant to herbicides.

mPing_EPSPS: Tests the TATSI system's ability to carry large-capacity DNA fragments. This version demonstrates the potential of the TATSI system for processing and inserting longer DNA fragments.

This study developed and validated the TATSI system. This innovative technology combines the advantages of transposase and CRISPR/Cas systems to significantly improve the efficiency and accuracy of DNA insertion. It can handle DNA fragments of various types and lengths, simplifies the operation process, and is suitable for a variety of plants. The TATSI system is of great significance in plant genome engineering and crop improvement.

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Reference

1. Liu, P., et al. Transposase-assisted target-site integration for efficient plant genome engineering. Nature (2024).