CRISPR screen and in-depth sequencing map DNA repair landscape stimulating prospects for genome editing

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Many powerful genome editing approaches must create breaks between the two strands of the DNA double helix and rely on the cell’s own DNA repair machinery to make the desired changes in the sequence. If a cell fails to repair these DNA double-stranded breaks (DSBs) efficiently and accurately, the genome becomes unstable and the cell dies.

Cells have developed two pathways to repair DSBs called non-homologous end junction (NHEJ) and homology-directed repair (HDR) with unique genetic components. Yet how a cell decides to fix DSBs and the factors involved remain unclear.

To enable a better understanding of DSB repair, scientists have now developed a new approach called Repair-seq that combines CRISPR-based genetic screens with deep site-specific DNA sequencing to profile the range of mutations produced on DNA sites targeted by different editions of the genome. tools. This new approach helps to understand how DNA repair pathways work to introduce programmable changes in the DNA sequence.

The study, conducted in the laboratories of Britt Adamson, PhD, assistant professor in the Department of Molecular Biology at Princeton, Jonathan Weissman, PhD, professor of biology at the Massachusetts Institute of Technology and researcher at Howard Hughes Medical Institute, and Cecilia Cotta-Ramusino , PhD, formerly at Editas Medicine and currently Vice President of Technology Development at Tessera Therapeutics, is published in an article in the journal Cell, titled “Mapping the genetic landscape of DNA double-strand break repair”.

Rodolphe Barrangou, PhD, professor emeritus of food, bioprocess and nutritional sciences at North Carolina State University and editor of The CRISPR journal, which is not involved in the study, comments: “This study critically addresses the second of two parts of genome editing: CRISPR made specific targeting and cleavage simple, so the main challenge now is to truly understand the diversity of repair pathways and outcomes that can be triggered.

“Repair-seq is a beautiful marriage of technological knowledge and biological knowledge,” said John Doench, PhD, research and development director of the Broad Institute’s Genetic Disruption Program, and who was not involved in the work. .

“We have long known that the mechanisms involved in repairing broken DNA are essential for genome editing, because to change the sequence of DNA, it must first be broken,” said Adamson. “But these processes are incredibly complex and therefore often difficult to disentangle.”

“Editing with double-stranded breaks has been the bread and butter of genome editing for a long time, but making intentional changes without unwanted mutations has been a huge challenge,” said Jeffrey Hussmann, PhD, lead author of study. “We set out to understand the mechanisms behind as many induced mutations as possible, believing that this could help us optimize the system.”

The researchers felt that simultaneously measuring the genetic determinants of a wide range of DSB repair outcomes would help studies of DSB repair. “With this goal in mind, we developed Repair-seq, a high-throughput method that combines deep locus-specific sequencing with genetic screens based on CRISPR interference (CRISPRi) to measure the effects of thousands of disturbances. genetic across the spectrum of mutations produced on targeted DNA lesions, ”the authors noted.

Repair-seq allows researchers to analyze the contributions of different pathways to repair specific DNA damage by simultaneously profiling how hundreds of individual genes affect mutations produced at damaged sites. Researchers can then generate mechanistic models of DNA repair and learn how these mechanisms impact genome editing.

Using Repair-seq, the team is studying the repair of DSBs induced by two programmable nucleases, Cas9 and Cas12a, generating edited sequence maps. “Systematic exploration of these maps isolated the processes responsible for common nuclease-induced mutations and revealed unexpected roles for canonical DNA damage response genes,” the authors noted.

The authors also applied Repair-seq to study DSB repair with different external repair templates (oligonucleotide donors). “This effort identified a range of genetically distinct processes by which model sequences are incorporated into breaks and demonstrated that Repair-seq is a flexible approach to discover how various genome editing technologies interact with endogenous repair processes.”

In two follow-up studies, the authors used Repair-Seq to identify the genetic determinants of basic and primary editing results, thereby helping to develop more efficient and accurate editing tools.

Analysis of data from Repair-seq experiments identified granular details in known repair pathways and identified new pathways that highlight the complexity involved in repairing double-strand breaks. The data discovered through this work is available to others who can use it to further study the genes and pathways involved in DNA repair.

“Together, these data demonstrate the broad utility of Repair-seq and provide a basis for further exploration of DNA repair pathways,” said the authors. Going forward, the team will continue to improve the platform and apply it to other genome editing technologies.

“We see Repair-seq as a tool that allows you to take a detailed picture of what genome editors are doing inside cells and then assess very quickly: ‘Is this a landscape that I can find design principles that will help improve the tool? ‘”said Adamson.” We are really excited to explore future applications. “

“These tools will be essential for mapping, understanding and possibly manipulating the repair pathways responsible for CRISPR-based editing results,” adds Barrangou. “As we move towards the clinical deployment of CRISPR technologies, we now need to fully verify the mosaicism of the edit results so that we can manipulate genomes in a precise and predictable manner. “


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