The Hujiazhi research team has developed new methods for optimizing gene editing and tracking DNA | Cell Discovery

Title: Optimizing genome editing strategy by primer-extension-mediated sequencing
Journal:
Jianhang Yin, Mengzhu Liu, Yang Liu, Jinchun Wu, Tingting Gan, Weiwei Zhang, Yinghui Li, Yaxuan Zhou, Jiazhi Hu
Published: 2019/03/26
Digital Identification Number: 10.1038/s4142 1-019-0088-8
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March 26, 2019, Peking University School of Life Sciences and Peking University-Tsinghua Joint Center for Life Sciences Hu Jiazhi Group published in
entitled Optimizing Genome Editing strategy by primer-extension-mediated sequencing paper. The study describes a new method that can be used to quantify both Cas9 editing efficiency and off-target activity, as well as the chromosomal abnormal structure caused by editing, or primer-extension-mediated sequencing (PEM-seq). This is a new technology that has a huge impact on areas such as gene editing and DNA damage repair.
CRISPR/Cas9 (clustered regular interspaced short palindromic repeats and CRISPR-associated proteins) is the most commonly used gene editing tool enzyme, widely used in scientific research, and clinical applications have stagnated due to its off-target activity. It anchors Cas9 to the target gene and induces DNA double-stranded fracture (DSB) by pairing it with a targeted DNA sequence through guide RNA (gRNA). In the process of gene editing induced DSB repair, there is a certain number of times that gene mutations or insertion of external DNA fragments will occur, so as to achieve the purpose of gene editing. In the practical application process, a good gene editing enzyme Cas9 needs to meet the three characteristics of high-efficiency cutting target point, low off-target activity and low chromosomal abnormality. At present, some PCR-based methods are used to estimate gene editing efficiency, but the reliability of the results needs to be improved;
Based on the principle of DNA double-stranded fracture and chromosomal sublocation, the Hujiazhi team developed PEM-seq, a new method with higher sensitivity and comprehensive and quantitative evaluation of gene editing, based on the existing high-volume sequencing method (Hu et al., Nature Protocols 2016). Compared with previous methods of evaluating Cas9 off-target activity based on second-generation sequencing, PEM-seq can not only sensitively identify Cas9 off-target points, but also accurately quantify crispr/Cas9 cutting efficiency at target points to find more efficient and safe Cas9 cutting site. At the same time, PEM-seq also reveals in depth the abnormal structure of chromosomes caused by gene editing near the target site, such as large fragment loss, chromosomal positional change, etc. Taking the RAG1A editing bits on the targeted treatment OF1 gene as an example, the Hujiazhi team found that there were abnormal structures such as a large number of chromosome missing inverts within 5kb of the Cas9 cutting bit 2.5% of the total editing events; These phenomena reveal the need for pre-application assessment of Cas9. The application of PEM-seq can comprehensively evaluate the cutting efficiency of Cas9 and most of the abnormal structure it causes, so as to optimize the gene editing strategy in order to achieve maximum editing efficiency and minimize off-target activity.At the same time, in order to reduce the off-target activity of CRISPR/Cas9, the Hujiazhi team combined the mutation site of the existing Cas9 variant, and through PEM-seq, a variant of FeCas9 with cutting efficiency comparable to that of wild Cas9 but with significantly lower off-target frequency was selected. The enzyme is used in a similar way to traditional Cas9.
addition, PEM-seq has great potential for genome stability research. Genome repair is closely related to dna repair and other factors, while traditional methods use PCR or molecular cloning to obtain repair information, sample size and preference. PEM-seq, similar to some previous high-volume sequencing methods, provides a larger amount of data and has a more obvious advantage, i.e. quantification. PEM-seq can gradually quantify the steps of DNA repair to detail the DNA process from injury to repair for a more accurate model.
summary: Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires requires engineered nucleases with high editingability low while off-targety. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
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(Source: Science.com)