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It is the dream
of many scientists to treat genetic diseases once and for all through the modification of the human genome.
The advent of a series of gene-editing systems based on CRISPR-Cas9 has given people hope
of turning dreams into reality.
At present, in vivo gene editing therapy based on the CRISPR-Cas9 system has been proof-of-concept, and the subsequent single-base editing system has also entered the clinical development stage
.
However, these systems still have limitations
in accurately inserting large pieces of DNA into the human genome.
Recently, a research team of scientists from the University of California, Berkeley and Stanford University has discovered a new series of large serine recombinases (LSRs).
These LSRs can precisely insert large DNA fragments over 7 kb into the human genome, providing a whole new set of tools for genome engineering research and the development of innovative therapies!
There are many causes of genetic diseases, sometimes just a "letter" on the gene encoding the protein is wrong, and in other cases, a large segment of the gene may be missing
.
Gene editing can restore cell function and treat disease
by precisely correcting single-base mutations or providing replacement copies of missing genes.
The early CRISPR-Cas9 system, which was able to precisely cut double-stranded DNA at specific locations in the human genome, activates the cell's DNA repair system, and if the correct DNA template is added at the same time, the cell may add the template to the
genome using a mechanism called homologous recombination.
WuXi AppTec
However, this method of gene editing is unreliable, sometimes resulting in a loss of bases near the site of double-stranded DNA breaks (DSBs) or random DNA fragment insertion
.
The new generation of base editing systems is able to change individual bases more precisely, but there are still limitations
in adding large DNA fragments.
"We want to develop a simple tool
that can add large pieces of DNA to the human genome.
" It does not require DNA double-strand breaks or rely on the cell's DNA repair machinery
.
Dr.
Patrick Hsu, a Berkeley researcher and co-founder of the newly established Arc Institute, one of the corresponding authors of the paper, said, "Our guess is that nature has evolved tools to solve these challenges, hiding in
bacteriophages and other mobile genetic elements.
“
that could add large DNA fragments to the human genome.
It does not require DNA double-strand breaks or rely on the cell's DNA repair machinery
.
Dr.
Patrick Hsu (Image source: Arc Institute)
Patrick Hsu (Image source: Arc Institute)
Dr.
Hsu studied under Dr.
Zhang Feng, a pioneer in the field of CRISPR, and made important contributions
to the development of the original CRISPR gene-editing system.
In the study, his team collaborated with scientists at Stanford University to study
LSRs present in bacteriophages.
LSR is an integrase carried by bacteriophages that integrates large pieces of DNA into the bacterial genome by recognizing specific sequences on the bacterial genome and phage DNA fragments
.
One advantage is that it can integrate DNA sequences containing thousands of base pairs, which can hold a complete human gene
.
"We think LSR has the potential to
deliver functional copies of mutated genes into the genome, restoring cellular function.
" Dr.
Hsu said
.
.
LSR can mediate the precise integration of large pieces of DNA into the bacterial genome (Image source: Reference [3])
LSR can mediate the precise integration of large pieces of DNA into the bacterial genome (Image source: Reference [3]) However, only a few LSRs have been used by scientists for genome editing, and one of the main reasons for limiting their application is the inefficient integration, with less than 10% of the genomes of cells modified
.
Instead of further modifying existing LSRs, the joint team decided to look for other LSRs
in nature's bacteriophages.
Over the long evolutionary journey of bacteria and bacteriophages, bacteriophages have evolved thousands of different LSRs that may provide more effective gene-editing tools
.
The researchers devised an algorithm that uses a computer system to search for DNA sequences from bacteriophages and bacteria to find signals
for new LSR species.
Using this algorithm, the researchers discovered more than 6,000 unique LSRs, expanding the number of known LSRs by more than 100 times!
Further research on these LSRs revealed that they have different characteristics, with some LSRs only recognizing specific sequences on the bacterial genome and integrating large DNA fragments into
sites containing specific sequences.
These sequences do not exist
in the human genome.
But artificially introducing these specific sequences, called "landing pads," into the human genome can accurately introduce large DNA fragments
.
Moreover, some newly discovered LSRs have better integration effect, in experiments, new LSRs can integrate DNA fragments of more than 7 kb into the genome of cells with 40~75% efficiency
.
.
Another class of sequences recognized by LSRs already exists
in the human genome.
This allows them to be used directly to introduce transgenes into the human genome and genetically engineer
.
The researchers note that these LSRs can be used for the production of CAR-T cells, and LSRs-mediated transgene integration can integrate transgenes into defined genomic loci and avoid the randomness of lentivirus-mediated transgene delivery
compared to current use of lentivirus-mediated transgene delivery.
And because different LSRs recognize different "landing zone" sequences, different LSRs can introduce multiple different transgenes into the same cell, further enhancing the function of
CAR-T cell therapy.
in the human genome.
This allows them to be used directly to introduce transgenes into the human genome and genetically engineer
.
Ultimately, the researchers identified more than 60 LSRs
that have been experimentally validated to insert large DNA fragments into human cells.
These LSRs do not require the involvement of any host intracellular proteins, do not require guidance RNA guidance, and their average molecular weight is only half that of Cas9 enzymes, making them easier to deliver into cells
.
that have been experimentally validated to insert large DNA fragments into human cells.
These LSRs do not require the involvement of any host intracellular proteins, do not require guidance RNA guidance, and their average molecular weight is only half that of Cas9 enzymes, making them easier to deliver into cells
.
The Arc team is currently developing LSRs that target the human genome and introduce therapeutic transgenes
into only one part of the human genome.
The goal of the scientists is to develop a DNA integration platform that can safely and efficiently integrate genes into a unique site in the human genome
, regardless of the genes that need to be integrated.
"This will provide a 'one-size-fits-all' gene therapy delivery strategy and be easier to scale up
.
" Dr.
Hsu said
.
, regardless of the genes that need to be integrated.
From the advent of CRISPR gene-editing tools, to the birth of single-base editing systems, lead editing systems, and the discovery of a new generation of Cas nucleases, the gene-editing toolbox has been growing to make it possible
to modify the human genome more accurately and efficiently.
The discovery of innovative LSRs adds a new tool to the gene editing toolbox, and we look forward to scientific breakthroughs to unleash the full potential of gene editing as soon as possible and transform it into a curative therapy
that benefits patients.
Resources:
Resources:
[1] Borrowing from bacterial viruses: New tools for large-scale genome
insertions.
Retrieved October 11, 2022, from
https://arcinstitute.
org/blog/genome_insertions?utm_source=twitter
Retrieved October 11, 2022, from https://arcinstitute.
org/blog/genome_insertions?utm_source=twitter
[2] Durrant et al.
, (2022).
Systematic discovery of recombinases for
efficient integration of large DNA sequences into the human genome.
Nature
Biotechnology, https://doi.
org/10.
1038/s41587-022-01494-w
, (2022).
Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome.
Nature Biotechnology, https://doi.
org/10.
1038/s41587-022-01494-w
[3] Stark (2017).
Making serine integrases work for us.
Current Opinion in
Microbiology, https://doi.
org/10.
1016/j.
mib.
2017.
04.
006
Making serine integrases work for us.
Current Opinion in Microbiology, https://doi.
org/10.
1016/j.
mib.
2017.
04.
006
