CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP–mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.
Get full access to this article
View all access options for this article.
References
1.
CongL, RanFA, CoxD, et al.Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339:819–823. DOI: 10.1126/science.1231143.
2.
MaliP, YangL, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science. 2013; 339:823–826. DOI: 10.1126/science.1232033.
3.
ZetscheB, Gootenberg JonathanS, Abudayyeh OmarO, et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163:759–771. DOI: 10.1016/j.cell.2015.09.038.
4.
SanderJD, JoungJK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotech. 2014; 32:347–355. DOI: 10.1038/nbt.2842.
5.
BrookhouserN, RamanS, PottsC, et al.May I cut in? Gene editing approaches in human induced pluripotent stem cells. Cells. 2017; 6. DOI: 10.3390/cells6010005.
6.
SuzukiK, TsunekawaY, Hernandez-BenitezR, et al.In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016; 540:144–149. DOI: 10.1038/nature20565.
7.
HeX, TanC, WangF et al.. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucl Acids Res. 2016; 44:e85. DOI: 10.1093/nar/gkw064.
8.
Schmid-BurgkJL, HöningK, EbertTS et al.. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat Commun. 2016; 7:12338. DOI: 10.1038/ncomms12338.
9.
OrthweinA, NoordermeerSM, WilsonMD et al.. A mechanism for the suppression of homologous recombination in G1 cells. Nature. 2015; 528:422–426. DOI: 10.1038/nature16142.
10.
IhryRJ, WorringerKA, SalickMR et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat Med. 2018; 24:939–946. DOI: 10.1038/s41591-018-0050-6.
11.
HaapaniemiE, BotlaS, PerssonJet al.CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018; 24:927. DOI: 10.1038/s41591-018-0049-z.
12.
FuY, FodenJA, KhayterCet al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotech. 2013; 31:822–826. DOI: 10.1038/nbt.2623.
13.
KosickiM, TombergK, BradleyA. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotech. 2018; 36:765–771. DOI: 10.1038/nbt.4192.
14.
KomorAC, KimYB, PackerMS et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016; 533:420–424. DOI: 10.1038/nature17946.
15.
KomorAC, ZhaoKT, PackerMS et al.Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv. 2017; 3:eaao4774. DOI: 10.1126/sciadv.aao4774.
16.
GajT, SirkSJ, BarbasCF. Expanding the scope of site-specific recombinases for genetic and metabolic engineering. Biotechnol Bioeng. 2014; 111:1–15. DOI: 10.1002/bit.25096.
17.
Standage-BeierK, WangX. Genome reprogramming for synthetic biology. Front Chem Sci Eng. 2017; 11:37–45. DOI: 10.1007/s11705-017-1618-2.
18.
GrindleyNDF, WhitesonKL, RicePA. Mechanisms of Site-Specific Recombination. Annu Rev Biochem. 2006; 75:567–605. DOI: 10.1146/annurev.biochem.73.011303.073908.
19.
BrafmanD, WillertK.Gene Transduction approaches in human embryonic stem cells. In: Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applications. (AtwoodG, ed.) IntechOpen, 2011. DOI: 10.5772/14163.
20.
St-PierreF, CuiL, PriestDG, et al.One-step cloning and chromosomal integration of DNA. ACS Synth Biol. 2013; 2:537–541. DOI: 10.1021/sb400021j.
21.
KarpinskiJ, HauberI, ChemnitzJ, et al.Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotech. 2016; 34:401–409. DOI: 10.1038/nbt.3467.
22.
AkopianA, HeJ, BoocockMR, et al.Chimeric recombinases with designed DNA sequence recognition. PNAS. 2003; 100:8688–8691. DOI: 10.1073/pnas.1533177100.
23.
MercerAC, GajT, FullerRP, et al.Chimeric TALE recombinases with programmable DNA sequence specificity. Nucl Acids Res. 2012:gks875. DOI: 10.1093/nar/gks875.
24.
ChaikindB, BessenJL, ThompsonDB, et al.A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucl Acids Res. 2016; 44:9758–9770. DOI: 10.1093/nar/gkw707.
ArnoldPH, BlakeDG, GrindleyND, et al.Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity. EMBO J. 1999; 18:1407–1414. DOI: 10.1093/emboj/18.5.1407.
27.
ProrocicMM, WenlongD, OlorunnijiFJ, et al.Zinc-finger recombinase activities in vitro. Nucl Acids Res. 2011; 39:9316–9328. DOI: 10.1093/nar/gkr652.
28.
YangW, SteitzTA. Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site. Cell. 1995; 82:193–207. DOI: 10.1016/0092-8674(95)90307-0.
29.
LiW, KamtekarS, XiongY, et al.Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science. 2005; 309:1210–1215. DOI: 10.1126/science.1112064.
30.
DiCarloJE, NorvilleJE, MaliP, et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucl Acids Res. 2013:gkt135. DOI: 10.1093/nar/gkt135.
31.
SikorskiRS, HieterP. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989; 122:19–27.
32.
EllisT, WangX, CollinsJJ. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotech. 2009; 27:465–471. DOI: 10.1038/nbt.1536.
33.
GuilingerJP, ThompsonDB, LiuDR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotech. 2014; 32:577–582. DOI: 10.1038/nbt.2909.
34.
NishimasuH, RanFA, HsuPD, et al.Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell. 2014; 156:935–949. DOI: 10.1016/j.cell.2014.02.001.
35.
Standage-BeierK, ZhangQ, WangX. Targeted large-scale deletion of bacterial genomes using CRISPR-nickases. ACS Synth Biol. 2015; 4:1217–1225. DOI: 10.1021/acssynbio.5b00132.
36.
GajT, SirkSJ, TingleRD et al.Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign. J Am Chem Soc. 2014; 136:5047–5056. DOI: 10.1021/ja4130059.
37.
NöllmannM, ByronO, StarkWM. Behavior of Tn3 resolvase in solution and its interaction with res. Biophys J. 2005; 89:1920–1931. DOI: 10.1529/biophysj.104.058164.
38.
HuJH, MillerSM, GeurtsMH, et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018; 556:57–63. DOI: 10.1038/nature26155.
39.
ChatterjeeP, JakimoN, JacobsonJM. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci Adv. 2018; 4:eaau0766. DOI: 10.1126/sciadv.aau0766.
40.
GordleyRM, SmithJD, GräslundT, et al.Evolution of programmable zinc finger-recombinases with activity in human cells. J Mol Biol. 2007; 367:802–813. DOI: 10.1016/j.jmb.2007.01.017.
41.
GersbachCA, GajT, GordleyRM et al.Directed evolution of recombinase specificity by split gene reassembly. Nucl Acids Res. 2010; 38:4198–4206. DOI: 10.1093/nar/gkq125.
42.
GajT, MercerAC, GersbachCA et al.Structure-guided reprogramming of serine recombinase DNA sequence specificity. PNAS. 2011; 108:498–503. DOI: 10.1073/pnas.1014214108.
43.
GajT, MercerAC, SirkSJ et al.A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucl Acids Res. 2013; 41:3937–3946. DOI: 10.1093/nar/gkt071.
44.
SirkSJ, GajT, JonssonAet al.Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants. Nucl Acids Res. 2014; 42:4755–4766. DOI: 10.1093/nar/gkt1389.
45.
WallenMC, GajT, IiiCFB. Redesigning recombinase specificity for safe harbor sites in the human genome. PLOS ONE. 2015; 10:e0139123. DOI: 10.1371/journal.pone.0139123.
BricknerJ.Genetic and epigenetic control of the spatial organization of the genome. Mol Biol Cell. 2017; 28:364–369. DOI: 10.1091/mbc.E16-03-0149.
48.
LiesnerR, ZhangW, NoskeN, et al.Critical amino acid residues within the φC31 integrase DNA-binding domain affect recombination activities in mammalian cells. Hum Gene Ther. 2010; 21:1104–1118. DOI: 10.1089/hum.2010.034.
49.
SiutiP, YazbekJ, LuTK. Synthetic circuits integrating logic and memory in living cells. Nat Biotech. 2013; 31:448–452. DOI: 10.1038/nbt.2510.
50.
YangL, NielsenAAK, Fernandez-RodriguezJ, et al.Permanent genetic memory with >1-byte capacity. Nat Meth. 2014; 11:1261–1266. DOI: 10.1038/nmeth.3147.
51.
WeinbergBH, PhamNTH, CaraballoLD, et al.Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat Biotech. 2017; 35:453–462. DOI: 10.1038/nbt.3805.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
