The CRISPR-Cas9 gene editing technology, which was introduced in 2012 and was awarded the Nobel Prize in Chemistry in 2020, has revolutionized the field of life sciences in just eight years. This technology has been widely applied in various areas of life science research and has even been used in clinical trials for the treatment of diseases like sickle cell anemia. Gene editing technology will continue to have a broad and profound impact on life science research, gene therapy, biotechnology, ethics, and more.

The principle of CRISPR-Cas9 technology involves two fundamental processes: guided RNA-directed Cas9 targeting of DNA cleavage and DNA repair. To avoid unintended side effects in gene therapy, it is crucial to develop gene editing techniques that are precise, controllable, and free from off-target effects. Understanding the results of DNA repair mediated by CRISPR-Cas9 editing is essential for the development of the technology. Previous studies have mainly focused on insertions/deletions as DNA repair outcomes induced by CRISPR-Cas9 editing in mammalian cells, while chromosome rearrangements and base substitutions have received less attention. Researchers led by Dr. Wang Shian and Dr. Li Fuli from Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) discovered a high frequency of base substitutions (point mutations) during gene editing in the yeast Rhodotorula mucilaginosa. They systematically analyzed and investigated the DNA repair outcomes and mechanisms mediated by CRISPR-Cas9, leading to the recent publication of their research in The CRISPR Journal, a specialized journal in gene editing.

Researchers identified 476 naturally repaired clones from 1.02 million transformed Red-paf1 yeast transformants of CRISPR-Cas9. Utilizing methods such as Sanger sequencing, Illumina sequencing, sequence structural variation analysis, chromosome karyotyping analysis, genetic mutation analysis, and statistical analysis, the DNA repair types were comprehensively analyzed, and the causes of repair were systematically investigated.

The study found that CRISPR-Cas9 gene editing-mediated DNA repair in Red-paf1 yeast exhibited diverse types, including DNA insertions/deletions, point mutations, chromosomal translocations, and short fragment reverse complementation, demonstrating various novel characteristics. Notably, DNA deletions of over 1 kb were not uncommon. Point mutations were non-randomly observed at specific target sites, distinct from the few identified base substitutions in HeLa cells and Saccharomyces cerevisiae. Chromosomal translocations occurred repetitively at two DNA breakage sites. Furthermore, the study revealed that point mutations and DNA deletions strongly relied on the NHEJ repair pathway genes Ku70, Ku80, Mre11, and RAD50; inactivation of any of these genes led to a sharp reduction in DNA repair capability. Additionally, point mutations and DNA deletions also depended on the error-prone DNA polymerase REV3 or Pol4; simultaneous inactivation of both REV3 and POL4 genes resulted in a noticeable decrease in DNA repair capability.

Although CRISPR-Cas9 gene editing technology has had a revolutionary impact, its application in the field of gene therapy still requires greater caution. Accurate gene editing remains challenging to achieve. While the research at the institute focuses on microorganisms, it also provides insights into gene editing in mammalian cells. Similarities have been found in the study, where a significant number of point mutations in human cancer cells are generated by error-prone DNA polymerases, and non-homologous end joining (NHEJ) is the main DNA repair mechanism in human cells. However, previous research did not sufficiently prioritize the characterization of natural DNA repair types, particularly point mutations, mediated by CRISPR-Cas9 in human cells. Further studies are needed to enhance our understanding of these natural DNA repair processes and ensure the safe application of gene editing technology in disease treatment.

Figure 1. DNA repair outcomes and mechanisms mediated by CRISPR-Cas9 in Red-paf1 yeast. A. Deletion repair; B. Base substitution (point mutation) repair; C. Repair type statistics at the CrtE site position 3; D. Identification of rare point mutations through deep sequencing; E. Chromosomal translocation identified through genome sequencing; F. Non-homologous end joining (NHEJ) pathway and CRISPR-Cas9-mediated DNA repair; G. Error-prone DNA polymerases and CRISPR-Cas9-mediated DNA repair; H. Model of DNA repair mechanism mediated by CRISPR-Cas9.

Graduate students Hong Jixuan, Meng Ziyue, and Zhang Ziqian are co-first authors of this work, while Researcher Wang Shian and Researcher Li Fuli are corresponding authors. This work was supported by the National Key Research and Development Program and the National Natural Science Foundation of China. (Text/Figure: Wang Shian, Meng Ziyue)

Original link: https://www.liebertpub.com/doi/10.1089/crispr.2021.0116

Jixuan Hong#, Ziyue Meng#, Zixi Zhang#, Hang Su, Yuxuan Fan, Ruilin Huang, Ruirui Ding, Ning Zhang, Fuli Li*, and Shi’an Wang*. Comprehensive analysis of CRISPR-Cas9 editing outcomes in yeastXanthophyllomyces dendrorhous.The CRISPR Journal, 2022, 10.1089/crispr.2021.0116.