systematically analyzes how genetic elements participate in DNA break repair. On the one hand, it helps people design more accurate and efficient gene editing tools. On the other hand, it helps people gain a deeper understanding of the DNA repair process, thereby designing new strategies to alleviate DNA instability and related tumors. and aging (1–3).
To this end, Princeton University Britt Adamson, MIT Jonathan Weissman, and Editas Medicine Company Cecilia Cotta-Ramusino and other researchers developed a new technology, Repair-seq, to knock down (CRISPRi) hundreds of DNA break repair-related genes in a high-throughput manner. And observe its impact on the "mutation spectrum" caused by Cas enzyme digestion of DNA double strands (3).
Repair-seq technology uses high-throughput monitoring of the "contribution" of DNA repair-related genes to double-strand break repair (3)
Researchers used this method to analyze the microhomology-mediated end joining of DNA through nearby sequences (Microhomology-mediated end The intrinsic mechanism of base deletion caused by joining); and unexpectedly found that seemingly similar base insertion mutations are regulated by different DNA non-homologous end joining (Non-Homologous End Joining (NHEJ)) key proteins.
Similar base insertion mutations after DNA double-strand breaks are regulated by different NHEJ key proteins (3)
The researchers further combined Repair-seq in the analysis of factors affecting Prime-editing efficiency and other work. It is expected that this method can be used to analyze various gene editing tools and other intrinsic mechanisms for DNA damage repair (3, 4).
This work was published in Cell on October 20, 2021.
Comments:
The "mutation spectrum" of DNA double-strand breaks in the article, this seemingly messy data, is beautifully displayed; it is a bit unexpected that the repeatability is so high, especially for the same site; maybe there are new rules. in.
However, as shown in the article, the "mutation spectrum" caused by different sites and different enzymes cutting ((cas9 or cas12a)) is still very different;
In addition, the article also mentioned that this technology cannot Capture all mutation types (such as large fragment loss, etc. cannot be captured);
In the future, with the advancement of single-cell genome technology and cost reduction, direct analysis of DNA double-strand breaks caused by high-energy rays will be more in line with physiological and pathological conditions. Source repair networks are a problem worth exploring, and the analysis of such high-dimensional data relies more on machine learning algorithms.
There are differences in the mutation spectrum of different restriction enzyme digestion (3)
restriction enzyme digestion of different sites "mutation spectrum" reproducibility decreases (3)
Corresponding author introduction:
https://www.badamsonlab.com/people
https://wi.mit. edu/people/member/weissman
https://www.tesseratherapeutics.com/team/cecilia-cotta-ramusino
References:
. P. A. Jeggo, L. H. Pearl, A. M. Carr, DNA repair, genome stability and cancer: a historical perspective. Nat Rev. Cancer. 16, 35–42 (2015).
. D. B. Lombard et al., DNA Repair, Genome Stability, and Aging. Cell. 120, 497–512 (2005).
. J. A. Hussmann et al., Mapping the genetic landscape of DNA double-strand break repair. Cell. 0(2021), doi:10.1016/J.CELL.2021.10.002.
4. P. J. Chen et al., Enhanced prime editing systems by manipulating cellular determinants of editing outcomes . Cell. 0 (2021), doi:10.1016/J.CELL.2021.09.018.
Original link:
https://www.cell.com/cell/fulltext/S0092-8674(21)01176-4
