Kun Xu
David Jay Segal
Zhiying Zhang- 1College of Animal Science and Technology, Northwest A&F University, Yangling, China
- 2Department of Biochemistry and Molecular Medicine, Genome Center, University of California, Davis, Davis, CA, United States
Editorial on the Research Topic
Precise Genome Editing Techniques and Applications
The CRISPR/Cas system, particularly CRISPR/Cas9 (Jinek et al., 2012; Cong et al., 2013), has been developed as a robust and versatile platform for manipulating the genomes of a variety of species. In recent years, numerous reports have suggested its powerful potential application for human gene therapy and life science research, as well as animal and plant breeding. This might be evidenced by the collections in this Research Topic, “Precise Genome Editing Techniques and Applications.”
Generally, the CRISPR/Cas9 nuclease is used to cleave target genomic DNA to generate site-specific double-strand breaks (DSBs), which are predominantly repaired via non-homologous end joining (NHEJ) or, to lesser extent, by homology-directed repair (HDR). The classical NHEJ repair pathway can generate small insertions or deletions (indels), resulting in loss-of-function of targeted coding genes by introducing a frameshift in the open reading frame (ORF). NHEJ mutagenesis is a highly popular strategy for gene manipulation. In addition to the classical NHEJ, alternative or accurate NHEJ-mediated repair can achieve precise genomic DNA deletions (Guo et al., 2018; Shou et al., 2018).
Two papers in this Research Topic by Chao et al. and Zhao et al. describe the manufacture of allele-specific knockout and double gene knockout mouse models for rapid disease gene validation and human xenograft studies, respectively. N6-methyladenosine (m6A) is a well-established epigenetic modification on eukaryotic mRNA. An increasing number of studies have uncovered the significance of m6A methylation, which has given rise the nascent field of “epitranscriptomics.” Another article in this volume (Huang et al.) describes a knock-out study in mouse spermatogonial GC-1 cells of the fat mass and obesity-associated (Fto) gene, which has been shown to act on the epitranscriptome as an m6A demethylase (Li et al., 2017; Lin et al., 2017).
On the other hand, the HDR repair pathway relies on homologous donor DNA to produce targeted gene knock-ins at the DSB site or gene replacement between two DSB sites. Precise point mutations and designed small indels can also be achieved by this method. One paper in this topic describes efforts to precisely correct the methyl-CpG binding protein 2 (MECP2) gene in the context of Rett syndrome (RTT) by CRISPR/Cas9-mediated HDR in human induced pluripotent stem cells (iPSCs). This report provides a reference for iPSC-based disease modeling and gene correction therapy (Le et al.).
Although the HDR-based genome can achieve gene insertions and precise substitutions, it is still confronted by several disadvantages during the precise editing process including low HDR efficiency, failure of biallelic targeting, complications of positive selection, and the re-deletion of selection markers.
It has been reported that inhibiting the key molecules of the competitive NHEJ pathway, such as DNA ligase IV (LIG4) and KU70, could improve the HDR efficiency effectively (Chu et al., 2015; Maruyama et al., 2015). We have previously developed a novel sgRNA-shRNA structure transcribing multiple sgRNAs for multiplex genome targeting (Yan et al., 2016). Here, the structure was further used for simultaneous LIG4 RNA interference and the enhanced HDR-based IGF2 SNP editing (Sun et al.). On the other hand, the HDR pathway can be also enhanced by the association of Cas9 with a variety of homologous recombination factors, such as yRad52 (Shao et al., 2017), dn53BP1 (Paulsen et al., 2017; Jayavaradhan et al., 2019), hRad51 (Rees et al., 2019), and CtIP (Tran et al.). A review paper in this topic (Liu et al.) further summarizes the methodologies and other considerations for improving the HDR efficiency.
Regarding biallelic targeting, we have previously reported a novel strategy using two donors with paired selectable markers (Wu et al., 2017). However, the removal of the selection is often required to allay concerns of marker-dependent effects. There are several “pop in and out” two-step techniques for marker-free genome engineering, including the Cre/LoxP system (Zhu et al., 2015), the piggyBac transposon (Xie et al., 2014), and the SSA repair mechanism (Li et al., 2018). This Research Topic presents a protocol article for biallelic HDR targeting using piggyBac-mediated selection removal (Jarazo et al.).
The ever-expanding repertoire of CRISPR editing systems includes the widely used Cas9 of Streptococcus pyogenes (SpCas9) (Jinek et al., 2012; Cong et al., 2013), as well as Streptococcus thermophilus (StCas9) (Xu et al., 2015), and Neisseria meningitides (NmCas9) (Hou et al., 2013). In addition, other proteins in the CRISPR family such as Cpf1/Cas12a (Zetsche et al., 2015) have been applied for genome editing. More recently, CRISPR/Cas-derived novel genome editing tools that do not create DSBs have been developed, including the cytidine and adenine base editors (CBE and ABE) (Komor et al., 2016; Gaudelli et al., 2017), as well as prime editors (PE) (Anzalone et al., 2019). The paper by Wu et al. in this topic describes efforts to increase the CBE scope and efficiency in rice.
The rapid development of genome editing technology has provided opportunities for modifying large animal models and domestic animal breeding. Pigs serve as an important agricultural resource as well as animal models for biomedical studies. In this topic, Yang and Wu summarize the genome editing of pigs in agricultural and biomedical applications. Off-target effects are one of the major concerns for genome editing research. The last two articles (Li et al.; Zhou et al.) in this Research Topic report no obvious off-target events in the offspring of genetically edited goats. However, it remains to be determined as to whether these observations might be affected by survivorship bias, as well as differences in off-target events between human gene therapy and animal genetic breeding.
In conclusion, the articles contained within this Research Topic illustrate the mechanisms and great potential of precise genome editing techniques to further scientific inquiry and produce useful outcomes that benefit society.
Author Contributions
KX wrote the manuscript draft. DS and ZZ supervised the topic and read the manuscript.
Funding
This work was supported by grants from the China Postdoctoral Science Foundation (2018T111111 and 2015M580887), the National Natural Science Foundation of China (NSFC, 31702099), and the National Transgenic Major Project of China (2018ZX08010-09B).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. doi: 10.1038/s41586-019-1711-4
Chu, V. T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K., et al. (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548. doi: 10.1038/nbt.3198
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. doi: 10.1126/science.1231143
Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., et al. (2017). Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471. doi: 10.1038/nature24644
Guo, T., Feng, Y.-L., Xiao, J.-J., Liu, Q., Sun, X.-N., Xiang, J.-F., et al. (2018). Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing. Genome Biol. 19:170. doi: 10.1186/s13059-018-1518-x
Hou, Z., Zhang, Y., Propson, N. E., Howden, S. E., Chu, L. F., Sontheimer, E. J., et al. (2013). Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl. Acad. Sci. U.S.A. 110, 15644–15649. doi: 10.1073/pnas.1313587110
Jayavaradhan, R., Pillis, D. M., Goodman, M., Zhang, F., Zhang, Y., Andreassen, P. R., et al. (2019). CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. Nat. Commun. 10:2866. doi: 10.1038/s41467-019-10735-7
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. doi: 10.1126/science.1225829
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., and Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424. doi: 10.1038/nature17946
Li, X., Bai, Y., Cheng, X., Kalds, P. G. T., Sun, B., Wu, Y., et al. (2018). Efficient SSA-mediated precise genome editing using CRISPR/Cas9. FEBS J 285, 3362–3375. doi: 10.1111/febs.14626
Li, Z., Weng, H., Su, R., Weng, X., Zuo, Z., Li, C., et al. (2017). FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase. Cancer Cell 31, 127–141. doi: 10.1016/j.ccell.2016.11.017
Lin, Z., Hsu, P. J., Xing, X., Fang, J., Lu, Z., Zou, Q., et al. (2017). Mettl3-/Mettl14-mediated mRNA N(6)-methyladenosine modulates murine spermatogenesis. Cell Res. 27, 1216–1230. doi: 10.1038/cr.2017.117
Maruyama, T., Dougan, S. K., Truttmann, M. C., Bilate, A. M., Ingram, J. R., and Ploegh, H. L. (2015). Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542. doi: 10.1038/nbt.3190
Paulsen, B. S., Mandal, P. K., Frock, R. L., Boyraz, B., Yadav, R., Upadhyayula, S., et al. (2017). Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing. Nat. Biomed. Eng. 1, 878–888. doi: 10.1038/s41551-017-0145-2
Rees, H. A., Yeh, W.-H., and Liu, D. R. (2019). Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nat. Commun. 10:2212. doi: 10.1038/s41467-019-09983-4
Shao, S., Ren, C., Liu, Z., Bai, Y., Chen, Z., Wei, Z., et al. (2017). Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int. J. Biochem. Cell Biol. 92, 43–52. doi: 10.1016/j.biocel.2017.09.012
Shou, J., Li, J., Liu, Y., and Wu, Q. (2018). Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-mediated nucleotide insertion. Mol. Cell 71, 498–509 e4. doi: 10.1016/j.molcel.2018.06.021
Wu, Y., Xu, K., Ren, C., Li, X., Lv, H., Han, F., et al. (2017). Enhanced CRISPR/Cas9-mediated biallelic genome targeting with dual surrogate reporter-integrated donors. FEBS Lett. 591, 903–913. doi: 10.1002/1873-3468.12599
Xie, F., Ye, L., Chang, J. C., Beyer, A. I., Wang, J., Muench, M. O., et al. (2014). Seamless gene correction of beta-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res. 24, 1526–1533. doi: 10.1101/gr.173427.114
Xu, K., Ren, C., Liu, Z., Zhang, T., Zhang, T., Li, D., et al. (2015). Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus. Cell Mol. Life Sci. 72, 383–399. doi: 10.1007/s00018-014-1679-z
Yan, Q., Xu, K., Xing, J., Zhang, T., Wang, X., Wei, Z., et al. (2016). Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure. Sci. Rep. 6:38970. doi: 10.1038/srep38970
Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771. doi: 10.1016/j.cell.2015.09.038
Keywords: precise genome editing, CRISPR, HDR efficiency, biallelic HDR targeting, off-target effect, animal model, base editing
Citation: Xu K, Segal DJ and Zhang Z (2020) Editorial: Precise Genome Editing Techniques and Applications. Front. Genet. 11:412. doi: 10.3389/fgene.2020.00412
Received: 19 February 2020; Accepted: 01 April 2020;
Published: 21 April 2020.
Edited and reviewed by: Youri I. Pavlov, University of Nebraska Medical Center, United States
Copyright © 2020 Xu, Segal and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: David Jay Segal, ZGpzZWdhbCYjeDAwMDQwO3VjZGF2aXMuZWR1; Zhiying Zhang, emhhbmd6aHkmI3gwMDA0MDtud2FmdS5lZHUuY24=