Studying concatenation of the Cas9-cleaved transgenes using barcodes

   In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9-linearized transgenes (RNP– experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP– experiment, six positive embryos were identified, four of which exhibited low-copy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2–4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.

1. Abe T., Inoue K., Furuta Y., Kiyonari H. Pronuclear microinjection during S-phase increases the efficiency of CRISPR-Cas9-assisted knockin of large DNA donors in mouse zygotes. Cell Rep. 2020; 31(7):107653. doi: 10.1016/j.celrep.2020.107653

2. Clarke R., Heler R., MacDougall M.S., Yeo N.C., Chavez A., Regan M., Hanakahi L., Church G.M., Marraffini L.A., Merrill B.J. Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Mol Cell. 2018;71(1):42-55.e8. doi: 10.1016/j.molcel.2018.06.005

3. Cock P.J.A., Antao T., Chang J.T., Chapman B.A., Cox C.J., Dalke A., Friedberg I., Hamelryck T., Kauff F., Wilczynski B., De Hoon M.J.L. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009;25(11):1422-1423. doi: 10.1093/bioinformatics/btp163

4. Cock P.J.A., Fields C.J., Goto N., Heuer M.L., Rice P.M. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2010;38(6): 1767-1771. doi: 10.1093/nar/gkp1137

5. Dai J., Cui X., Zhu Z., Hu W. Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos. Int J Biol Sci. 2010;6(7):756-768. doi: 10.7150/ijbs.6.756

6. Danner E., Lebedin M., De La Rosa K., Kühn R. A homology independent sequence replacement strategy in human cells using a CRISPR nuclease. Open Biol. 2021;11(1):200283. doi: 10.1098/rsob.200283

7. Gurtan A.M., Lu V., Bhutkar A., Sharp P.A. In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs. RNA. 2012;18(6):1116-1122. doi: 10.1261/rna.032680.112

8. Harms D.W., Quadros R.M., Seruggia D., Ohtsuka M., Takahashi G., Montoliu L., Gurumurthy C.B. Mouse genome editing using the CRISPR/Cas system. Curr Protoc Hum Genet. 2014;83:15.7.1-15.7.27. doi: 10.1002/0471142905.hg1507s83

9. Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C., Hauer M., Zhou K., Lin S., Kaplan M., Iavarone A.T., Charpentier E., Nogales E., Doudna J.A. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014;343(6176):1247997. doi: 10.1126/science.1247997

10. Maggio I., Holkers M., Liu J., Janssen J.M., Chen X., Gonçalves M.A.F.V. Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci Rep. 2014;4(1):5105. doi: 10.1038/srep05105

11. Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826. doi: 10.1126/science.1232033

12. Maltseva E.A., Vasil’eva I.A., Moor N.A., Kim D.V., Dyrkheeva N.S., Kutuzov M.M., Vokhtantsev I.P., Kulishova L.M., Zharkov D.O., Lavrik O.I. Cas9 is mostly orthogonal to human systems of DNA break sensing and repair. PLoS One. 2023;18(11):e0294683. doi: 10.1371/journal.pone.0294683

13. Nishimasu H., Ran F.A., Hsu P.D., Konermann S., Shehata S.I., Dohmae N., Ishitani R., Zhang F., Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5): 935-949. doi: 10.1016/j.cell.2014.02.001

14. Reginato G., Dello Stritto M.R., Wang Y., Hao J., Pavani R., Schmitz M., Halder S., Morin V., Cannavo E., Ceppi I., Braunshier S., Acharya A., Ropars V., Charbonnier J.-B., Jinek M., Nussenzweig A., Ha T., Cejka P. HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing. Nat Commun. 2024;15(1):5789. doi: 10.1038/s41467-024-50080-y

15. Richardson C.D., Ray G.J., DeWitt M.A., Curie G.L., Corn J.E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016;34(3):339-344. doi: 10.1038/nbt.3481

16. Rohan R.M., King D., Frels W.I. Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes. Nucleic Acids Res. 1990;18(20):6089-6095. doi: 10.1093/nar/18.20.6089

17. Sakuma T., Nakade S., Sakane Y., Suzuki K.-I.T., Yamamoto T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc. 2016;11(1):118-133. doi: 10.1038/nprot.2015.140

18. Schimmel J., Kool H., Van Schendel R., Tijsterman M. Mutational signatures of non‐homologous and polymerase theta‐mediated end-joining in embryonic stem cells. EMBO J. 2017;36(24):3634-3649. doi: 10.15252/embj.201796948

19. Smirnov A., Fishman V., Yunusova A., Korablev A., Serova I., Skryabin B.V., Rozhdestvensky T.S., Battulin N. DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote. Nucleic Acids Res. 2020; 48(2):719-735. doi: 10.1093/nar/gkz1085

20. Stephenson A.A., Raper A.T., Suo Z. Bidirectional degradation of DNA cleavage products catalyzed by CRISPR/Cas9. J Am Chem Soc. 2018;140(10):3743-3750. doi: 10.1021/jacs.7b13050

21. Suzuki K., Tsunekawa Y., Hernandez-Benitez R., Wu J., Zhu J., Kim E.J., Hatanaka F., Yamamoto M., Araoka T., Li Z., Kurita M., Hishida T., Li M., Aizawa E., Guo S., Chen S., Goebl A., Soligalla R.D., Qu J., Jiang T., Fu X., Jafari M., Esteban C.R., Berggren W.T., Lajara J., Nuñez-Delicado E., Guillen P., Campistol J.M., Matsuzaki F., Liu G.-H., Magistretti P., Zhang K., Callaway E.M., Zhang K., Belmonte J.C.I. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016; 540(7631):144-149. doi: 10.1038/nature20565

22. Taheri-Ghahfarokhi A., Taylor B.J.M., Nitsch R., Lundin A., Cavallo A.-L., Madeyski-Bengtson K., Karlsson F., Clausen M., Hicks R., Mayr L.M., Bohlooly-Y.M., Maresca M. Decoding non-random mutational signatures at Cas9 targeted sites. Nucleic Acids Res. 2018; 46(16):8417-8434. doi: 10.1093/nar/gky653

23. Takeo T., Nakagata N. Combination medium of cryoprotective agents containing l-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm. Lab Anim. 2010;44(2):132-137. doi: 10.1258/la.2009.009074

24. Takeo T., Nakagata N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod. 2011;85(5):1066-1072. doi: 10.1095/biolreprod.111.092536

25. Takeo T., Hoshii T., Kondo Y., Toyodome H., Arima H., Yamamura K., Irie T., Nakagata N. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol Reprod. 2008;78(3): 546-551. doi: 10.1095/biolreprod.107.065359