![]() This turned out to be an asset because it introduced editing flexibility. Building the serine recombinase platformīut it wasn’t simple: many of the recombinases they discovered integrate payloads into a short, specific attachment site sequence that is not found in the human genome. Overall, their large-scale discovery effort expanded the diversity of the LSR enzyme family by over 100-fold, resulting in a huge library of enzyme variants that offer a wide variety of options to edit different locations in the human genome. The team devised a clever computational approach to search bacterial and bacteriophage genome sequences for hints of new LSR enzymes and to map their attachment sites, gathering the critical pieces of information necessary to coax the LSR to insert DNA into a desired site for genome editing. According to Tycko, “we imagined that if we could discover new LSRs at a large scale, we would find some that already have robust specificity and efficiency for human genome targeting, right out of the box.” Owing to their long evolutionary battle over billions of years fought through genetic warfare, bacteria and bacteriophages are a treasure trove of enzymes that detect, modify, cut, and combine DNA and RNA, both offensively and defensively. Only a handful of LSRs have previously been characterized and developed into research tools, and they have such low efficiency that their utility is greatly limited. Because LSRs can insert many thousands of nucleotides-big enough to supply an entire human gene, or even a couple genes-“we saw the potential of LSR tools to deliver a functional version of a mutated gene into the human genome to restore cellular function,” says Hsu. “This avoids the imprecise smashing of the genetic keyboard at the cut site during DNA repair,” explains co-first author Josh Tycko of Stanford University. Through some DNA acrobatics, LSRs integrate DNA sequences without ever creating a double-strand DNA break in the genome. These LSR enzymes integrate DNA into a bacterial genome by matching pre-existing DNA sequence tags known as attachment sites on both the payload and the target genomic site. “They had naturally evolved to tackle the exact problem we were hoping to solve”, says co-first author and Arc senior scientist Matthew Durrant. In the natural world, bacterial viruses known as bacteriophages carry LSR enzymes that integrate large stretches of bacteriophage DNA into new host bacterial genomes, thereby facilitating viral spread. Working with collaborators from Stanford University-Ami Bhatt, Lacramioara Bintu, and Michael Bassik-Hsu’s team turned to microbial evolutionary biology to study large serine recombinases (LSRs). “We reasoned that nature may have already evolved solutions to these challenges through bacteriophages and other mobile genetic elements, perhaps the oldest systems for genetic diversification.” Hunting for new genome editing systems in bacterial genomes “We wanted a simple tool for inserting large DNA sequences into a human genome that wouldn't require double-stranded DNA breaks or rely on cellular DNA repair machinery,” says Arc Institute and UC Berkeley Bioengineering investigator Patrick Hsu. Newer variations on CRISPR, such as base or prime editing, are more precise but are limited to making only small changes. However, this gene editing approach is unreliable, often resulting in random insertions or deletions of several nucleotide letters at the cut site. If you supply an additional, corrective DNA template along with a CRISPR cut, the cell will sometimes patch up the DNA break by inserting the template. This causes a double-stranded DNA break, which activates the cell to stitch its DNA back together. The original version of CRISPR, known as CRISPR-Cas9, uses a kind of molecular scissors in order to cut the human genome at a desired location. Editing the genome-either by precisely correcting the mutation or by supplying a replacement version of the missing or damaged gene-could rescue cellular function and alleviate disease. Genetic diseases can result from small “typos” or entire missing “paragraphs” in our 3 billion-letter genome. Today, Arc Institute scientists report in Nature Biotechnology the discovery of new serine recombinases, a family of genome editing tools, for precisely inserting large DNA payloads into the human genome. Electron micrograph of multiple bacteriophages attached to a bacterial cell wall.
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