Table 2.

In vivo epigenome editing and transcriptional modulation using CRISPR technology

Purpose	CRISPR strategy	Delivery method	Significance	Animal	Reference
DNA methylation	targeted CpG methylation using dCas9-MQ1 (a fusion between dCas9 and prokaryotic DNA methyltransferase MQ1)	induce targeted CpG methylation in mice by zygote microinjection of dCas9-MQ1	specific, rapid and efficient strategy to achieve locus- specific cytosine modifications in the genome in vivo	mouse embryo	(Lei et al. 2017)
DNA demethylation	dCas9-peptide repeat was used to recruit multiple copies of scFv-TET1 catalytic domain fusion for amplification of DNA demethylation	in utero electroporation of all-in-one vectors	in vivo targeted demethylation in the brain of mouse fetuses	mouse fetus	(Morita et al. 2016)
gene activation	induce trans-epigenetic remodeling by co-introducing a truncated 14bp guide RNA (dgRNA) and an MS2- P65-HSF1 (MPH) transcriptional activation complex into Cas9- or dCas9-expressing mice	AAV9 delivery of dgRNA and MPH via intramuscular, facial vein, intra-cerebral, and tail-vein injections	ameliorate disease phenotypes for type I diabetes, acute kidney injury, and muscular dystrophy	adult mouse	(Liao et al. 2017)
gene activation	a Cre-dependent SunTag-p65-HSF1 (SPH) transgenic mouse that stably expressing dCas9-10xGCN4 of SunTag and p65-HSF1 of SAM	AAV8 delivery of Cre and sgRNAs via stereotactic injection	directly converted astrocytes into functional neurons in the brain by activating endogenous neurogenic genes	adult mouse	(Zhou et al. 2018)
gene activation	a split Cas9 AAV system, which Cas9 N-terminal lobe fused to the N- split intein (Cas9N) while the C- terminal lobe fused to the C-split intein (Cas9C); Cas9C was then fused to the tripartite VPR transactivation domain	AAV9 delivery of Cas9C- VPR and Cas9N-gRNAs via intramuscular injection	enable dual AAV delivery of Cas9-VPR fusion proteins for gene activation in the muscle	adult mouse	(Chew et al. 2016)
gene activation	dCas9-VPR system coupled with Gal4-UAS activation to induce dominant phenotypes in vivo	flies of the genotype dCas9-VPR were crossed to homozygous sgRNA flies	first demonstration of dCas9-based activation in a multicellular animal	Drosop hila	(Lin et al. 2015)
gene activation	dCas9-VPR system coupled with a genome-wide collection of flies expressing sgRNAs	flies of the genotype dCas9-VPR were crossed to a collection of flies expressing sgRNAs	generated strong gain-of- function phenotypes in multiple tissues in vivo for large-scale genetic screens	Drosop hila	(Ewen- Campen et al. 2017)
gene activation	a flySAM1.0 system consisted of dCas9-VP64, MCP-p65-HSF1 and sgRNA-luc was used for in vivo CRISPRa luciferase assay; a flySAM2.0 all-in-one system was used for tissue-specific CRISPRa with a single genetic cross	flySAM2.0 lines were crossed to a Gal4 line	improve potency, scalability, and ease of use for systematic overexpression genetic analysis and screens	Drosop hila	(Jia et al. 2018)
gene activation	a Cre-inducible CRISPRa system (dCas9-SunTag) for hepatocyte- specific gene activation in the liver	AAV delivery of Cre in dCas9-expressing mice, followed by hydrodynamic injection of plasmid pools (Fah, TA and gRNA)	enable parallel and combinatorial genetic screening for drivers and suppressors of tumor initiation and proliferation in live animals	adult mouse	(Wangenst een et al. 2017)
gene activation	an optogenetic far-red light (FRL)- activated CRISPR-dCas9 system (FACE) based on dCas9, hybrid transactivator MS2-p65-HSF1, the bacterial phytochrome BphS and FRL illumination; in the presence of FRL, BphS is activated to convert GTP into c-di-GMP. Increased cytosolic c- di-GMP production dimerizes p65- VP64-NLS-BldD to induce expression of MS2-p65-HSF1, which are further recruited by the MS2 box of the sgRNA-dCas9 complex to activate target gene expression.	plasmid electroporation of FACE system into mouse muscles, followed by illumination with FRL	promote differentiation of induced pluripotent stem cells into functional neurons via precise spatiotemporal control of endogenous gene expression and trans- epigenetic remodeling	adult mouse	(Shao et al. 2018)
gene activation and repression	dCas9-VP64 mediated transcriptional perturbation	tail-vein injection of B- ALL cells expressing dCas9-VP64 and a custom sgRNA library	interrogate tumor phenotypes in vivo for modeling cancer progression and therapeutic relapse	adult mouse	(Braun et al. 2016)
gene activation and repression	a modular dual-AAV split-dCas9 system consisting of dCas9-based transcriptional regulation modules (VP64, RTA and p65 activators) (KRAB, DNMT3A, DNMT3L or FOG1 repressor)	AAV delivery of AAV- split-KRAB-dCas9-Nrl into mouse retina	enable in situ gene therapy by targeting disease in a genomically scarless and reversible manner	adult mouse	(Moreno et al. 2018)
gene silencing	a dual-vector AAV8 system, which two AAV vectors separately express a dSaCas9-KRAB repressor and a sgRNA	AAV8 delivery of a dSaCas9-KRAB and a sgRNA via tail-vein injection	reduce serum Pcsk9 and cholesterol levels in the liver	adult mouse	(Thakore et al. 2018)
gene silencing	dCas9-KRAB with a pseudotarget fishing strategy to achieve superior targeting specificity without detectable off-target activity	lentiviral delivery of dCas9-KRAB-U6-sgRNA via stereotactic injection	enable rapid and accurate gene function investigation in the mammalian brain	adult mouse	(Zheng et al. 2018)
histone demethylation, gene activation and repression	dCas9-LSD1 demethylase, dCas9- KRAB repressor, or dCas9-VP64 that incorporates the synergistic activation mediator (SAM) system	electroporation of a dCas9- effector and multiple sgRNAs	probe gene regulatory interactions during early neural crest development	chicken embryo	(Williams et al. 2018)

Note: Fah, fumarylacetoacetate hydrolase; HSF1, heat shock factor 1; KRAB, Krüppel associated box; LSD1, lysine-specific histone demethylase 1A; MS2, viral RNA stem-loop motifs; Pcsk9, proprotein convertase subtilisin/kexin type 9; SAM, synergistic activation mediator; scFv, single-chain variable fragment; TA, transcriptional activator; TET1, methylcytosine dioxygenase 1; UAS, upstream activating sequence; VPR, VP64-p65-Rta.