Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
13 May 2024
13 May 2024
Historique:
received:
12
04
2023
accepted:
10
04
2024
medline:
14
5
2024
pubmed:
14
5
2024
entrez:
13
5
2024
Statut:
aheadofprint
Résumé
Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.
Identifiants
pubmed: 38740992
doi: 10.1038/s41587-024-02238-8
pii: 10.1038/s41587-024-02238-8
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : 393547839
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : INST 247/870-1 FUGG
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : 402733153
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : 455784893
Informations de copyright
© 2024. The Author(s).
Références
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
pubmed: 23287718
pmcid: 3795411
doi: 10.1126/science.1231143
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, 2579–2586 (2012).
doi: 10.1073/pnas.1208507109
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
doi: 10.1126/science.1225829
pubmed: 22745249
pmcid: 6286148
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
pubmed: 23287722
pmcid: 3712628
doi: 10.1126/science.1232033
Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023).
pubmed: 36656942
doi: 10.1126/science.add8643
van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016).
pubmed: 27499295
doi: 10.1016/j.molcel.2016.06.037
Chakrabarti, A. M. et al. Target-specific precision of CRISPR-mediated genome editing. Mol. Cell 73, 699–713 (2019).
pubmed: 30554945
pmcid: 6395888
doi: 10.1016/j.molcel.2018.11.031
Taheri-Ghahfarokhi, A. et al. Decoding non-random mutational signatures at Cas9 targeted sites. Nucleic Acids Res. 46, 8417–8434 (2018).
pubmed: 30032200
pmcid: 6144780
doi: 10.1093/nar/gky653
Shen, M. W. et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563, 646–651 (2018).
pubmed: 30405244
pmcid: 6517069
doi: 10.1038/s41586-018-0686-x
Allen, F. et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat. Biotechnol. 37, 64–82 (2019).
doi: 10.1038/nbt.4317
Leenay, R. T. et al. Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells. Nat. Biotechnol. 37, 1034–1037 (2019).
pubmed: 31359007
pmcid: 7388783
doi: 10.1038/s41587-019-0203-2
Chen, W. et al. Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair. Nucleic Acids Res. 47, 7989–8003 (2019).
pubmed: 31165867
pmcid: 6735782
doi: 10.1093/nar/gkz487
Molla, K. A. & Yang, Y. Predicting CRISPR/Cas9-induced mutations for precise genome editing. Trends Biotechnol. 38, 136–141 (2020).
pubmed: 31526571
doi: 10.1016/j.tibtech.2019.08.002
Lemos, B. R. et al. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. Proc. Natl Acad. Sci. USA 115, E2010–E2047 (2018).
doi: 10.1073/pnas.1716855115
Shi, X. et al. Cas9 has no exonuclease activity resulting in staggered cleavage with overhangs and predictable di- and tri-nucleotide CRISPR insertions without template donor. Cell Discov. 5, 53 (2019).
pubmed: 31636963
pmcid: 6796948
doi: 10.1038/s41421-019-0120-z
Shou, J., Li, J., Liu, Y. & Wu, Q. Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-mediated nucleotide insertion. Mol. Cell 71, 498–509 (2018).
pubmed: 30033371
doi: 10.1016/j.molcel.2018.06.021
Gisler, S. et al. Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency. Nat. Commun. 10, 1598 (2019).
Jones Jr, S. K. et al. Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nat. Biotechnol. 39, 84–93 (2021).
Lazzarotto, C. R. et al. CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity. Nat. Biotechnol. 38, 1317–1327 (2020).
pubmed: 32541958
pmcid: 7652380
doi: 10.1038/s41587-020-0555-7
Kim, D. & Kim, J.-S. DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA. Genome Res. 28, 1894–1900 (2018).
pubmed: 30413470
pmcid: 6280750
doi: 10.1101/gr.236620.118
Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).
pubmed: 28459458
pmcid: 5924695
doi: 10.1038/nmeth.4278
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–198 (2015).
pubmed: 25513782
doi: 10.1038/nbt.3117
Ivanov, I. E. et al. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. Proc. Natl Acad. Sci. USA 117, 5853–5860 (2020).
pubmed: 32123105
pmcid: 7084090
doi: 10.1073/pnas.1913445117
Pacesa, M. et al. Structural basis for Cas9 off-target activity. Cell 185, 4067–4081 (2022).
pubmed: 36306733
pmcid: 10103147
doi: 10.1016/j.cell.2022.09.026
1000 Genomes Project Consortiumet al. A global reference for human genetic variation. Nature 526, 68–74 (2015).
doi: 10.1038/nature15393
Cancellieri, S. et al. Human genetic diversity alters off-target outcomes of therapeutic gene editing. Nat. Genet. 138, 3993–3993 (2022).
Scott, D. A. & Zhang, F. Implications of human genetic variation in CRISPR-based therapeutic genome editing. Nat. Med. 23, 1095–1101 (2017).
pubmed: 28759051
pmcid: 5749234
doi: 10.1038/nm.4377
Lessard, S. et al. Human genetic variation alters CRISPR–Cas9 on- and off-targeting specificity at therapeutically implicated loci. Proc. Natl Acad. Sci. USA 114, E112157–E11266 (2017).
Krysler, A. R., Cromwell, C. R., Tu, T., Jovel, J. & Hubbard, B. P. Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences. Nat. Commun. 13, 1617 (2022).
pubmed: 35338140
pmcid: 8956631
doi: 10.1038/s41467-022-29202-x
Zook, J. M. et al. An open resource for accurately benchmarking small variant and reference calls. Nat. Biotechnol. 37, 561–566 (2019).
pubmed: 30936564
pmcid: 6500473
doi: 10.1038/s41587-019-0074-6
Zook, J. M. et al. Extensive sequencing of seven human genomes to characterize benchmark reference materials. Sci. Data 3, 160025 (2016).
pubmed: 27271295
pmcid: 4896128
doi: 10.1038/sdata.2016.25
Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).
pubmed: 30082871
pmcid: 6107069
doi: 10.1038/s41591-018-0137-0
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
pubmed: 29512652
pmcid: 5951633
doi: 10.1038/nature26155
Lee, J. K. et al. Directed evolution of CRISPR–Cas9 to increase its specificity. Nat. Commun. 9, 3048 (2018).
Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).
pubmed: 28931002
pmcid: 5918688
doi: 10.1038/nature24268
Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).
pubmed: 29431739
pmcid: 6066108
doi: 10.1038/nbt.4066
Schmid-Burgk, J. L. et al. Highly parallel profiling of Cas9 variant specificity. Mol. Cell 78, 794–800.e8 (2020).
pubmed: 32187529
pmcid: 7370240
doi: 10.1016/j.molcel.2020.02.023
Zeharia, A. et al. Acute infantile liver failure due to mutations in the TRMU gene. Am. J. Hum. Genet. 85, 401–407 (2009).
pubmed: 19732863
pmcid: 2771591
doi: 10.1016/j.ajhg.2009.08.004
Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 364, 286–289 (2019).
pubmed: 31000663
pmcid: 6589096
doi: 10.1126/science.aav9023
Atkins, A. et al. Off-target analysis in gene editing and applications for clinical translation of CRISPR/Cas9 in HIV-1 therapy. Front. Genome Ed. 3, 673022 (2021).
pubmed: 34713260
pmcid: 8525399
doi: 10.3389/fgeed.2021.673022
Cameron, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 14, 600–606 (2017).
pubmed: 28459459
doi: 10.1038/nmeth.4284
Kim, D. et al. Digenome-Seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
pubmed: 25664545
doi: 10.1038/nmeth.3284
Zuo, Z. & Liu, J. Cas9-catalyzed DNA cleavage generates staggered ends: evidence from molecular dynamics simulations. Sci. Rep. 5, 37584 (2016).
Schep, R. et al. Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance. Mol. Cell 81, 2216–2230 (2021).
pubmed: 33848455
pmcid: 8153251
doi: 10.1016/j.molcel.2021.03.032
Xue, C. & Greene, E. C. DNA repair pathway choices in CRISPR–Cas9-mediated genome editing. Trends Genet. 37, 639–656 (2021).
pubmed: 33896583
pmcid: 8187289
doi: 10.1016/j.tig.2021.02.008
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
pubmed: 32572269
doi: 10.1038/s41587-020-0561-9
Zhao, Z., Shang, P., Mohanraju, P. & Geijsen, N. Prime editing: advances and therapeutic applications. Trends Biotechnol. 41, 1000–1012 (2023).
pubmed: 37002157
doi: 10.1016/j.tibtech.2023.03.004
Fiumara, M. et al. Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01915-4 (2023)
Jiang, F. et al. Structures of a CRISPR–Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).
pubmed: 26841432
pmcid: 5111852
doi: 10.1126/science.aad8282
Hennig, B. P. et al. Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol. G3 (Bethesda) 8, 79–89 (2018).
pubmed: 29118030
doi: 10.1534/g3.117.300257
Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014).
pubmed: 25079858
pmcid: 4248319
doi: 10.1101/gr.177881.114
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at arXiv https://doi.org/10.48550/arXiv.1303.3997 (2013).
Lawrence, M. et al. Software for computing and annotating genomic ranges. PLoS Comput. Biol. 9, e1003118 (2013).
pubmed: 23950696
pmcid: 3738458
doi: 10.1371/journal.pcbi.1003118
Huber, W. et al. Orchestrating high-throughput genomic analysis with bioconductor. Nat. Methods 12, 115–121 (2015).
pubmed: 25633503
pmcid: 4509590
doi: 10.1038/nmeth.3252
Rashmi, K. V. & Gilad-Bachrach, R. DART: dropouts meet multiple additive regression trees. Preprint at arXiv https://doi.org/10.48550/arXiv.1505.01866 (2015).
Wagih, O. ggseqlogo: a versatile R package for drawing sequence logos. Bioinformatics 33, 3645–3647 (2017).
pubmed: 29036507
doi: 10.1093/bioinformatics/btx469
Lowy-Gallego, E. et al. Variant calling on the GRCh38 assembly with the data from phase three of the 1000 Genomes Project. Wellcome Open Res. 4 https://doi.org/10.12688/wellcomeopenres.15126.2 (2019).
Cunningham, F. et al. Ensembl 2022. Nucleic Acids Res. 50, D988–D995 (2022).
pubmed: 34791404
doi: 10.1093/nar/gkab1049
Papapetrou, E. P. & Sadelain, M. Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector. Nat. Protoc. 6, 1251–1273 (2011).
pubmed: 21886095
doi: 10.1038/nprot.2011.374
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
pubmed: 24695404
pmcid: 4103590
doi: 10.1093/bioinformatics/btu170
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
pubmed: 30809026
pmcid: 6533916
doi: 10.1038/s41587-019-0032-3
Yau, E. H. & Rana, T. M. Next-generation sequencing of genome-wide CRISPR screens. Methods Mol. Biol. 1712, 203–216 (2018).
pubmed: 29224076
pmcid: 6089254
doi: 10.1007/978-1-4939-7514-3_13
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).
pubmed: 25357182
doi: 10.1038/nbt.3081
Longo, G. M. C. et al. BreakTag links CRISPR/Cas9 double-strand break profile to gene editing precision. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223772 (2024).
Longo, G. M. C. et al. BreakTag links CRISPR/Cas9 double-strand break profile to gene editing precision. https://github.com/roukoslab/breaktag (2024).
Longo, G. M. C. et al. BreakTag links CRISPR/Cas9 double-strand break profile to gene editing precision. https://github.com/roukoslab/breakinspectoR (2024).