rna - guided machineries 
 provide highly selective and rationally 
 programmable tools for the site - specific manipulation of nucleic acids . several endogenous riboproteins are known that are steered toward 
 their endogenous targets by nucleic acid hybridization and that are 
 readily re - addressed toward new targets by expression or administration 
 of artificial external guidernas . those include the snorna - guided 
 2-o - methylation and pseudo - uridinylation machineries 
 and the microrna - guided rna - induced silencing complex . the harnessing 
 of the latter machinery , better known as rna interference , has developed into a standard tool in cell biology . besides harnessing endogenous eukaryotic machineries , the engineering 
 of artificial riboproteins for the site - specific manipulation of nucleic 
 acids comes more and more into focus now . tools are highly desired 
 that simplify genetic engineering and 
 that help to elucidate the role of point mutations and rna modifications . besides their application in basic biology research , such tools 
 have potential for translation into individualized medicine . a highly 
 topical example is the re - engineering of the bacterial crispr - cas9 
 system for site - selective genome editing in eukaryotic cells . endogenous riboproteins are typically assembled 
 by molecular recognition 
 between specific protein and rna structures . the formation of a single covalent bond between an rna and a protein 
 component , however , is virtually unknown for that purpose . nevertheless , 
 we could recently demonstrate the assembly and functioning of highly 
 selective adenosine ( a)-to - inosine rna editing machineries inside 
 living cells following the latter approach . since inosine is biochemically read as guanosine ( g ) , editing formally 
 creates a - to - g point mutations at the rna level . if rna editing is 
 directed to the open reading frame , 12 out of the 20 canonical amino 
 acids can be substituted , including most 
 of the polar residues essential for enzyme catalysis , post - translational 
 protein modification , or signaling . furthermore , editing in the non - coding 
 part of the rna can interfere with translation initiation ( start codon ) , 
 translation stop , microrna action , and splicing among others . thus , the potential of site - directed rna editing for application 
 in basic biology research and medicine is evident . we apply the snap - tag technology to 
 assemble the editing machinery via covalent bond formation . this technology 
 requires the fusion of a snap - tag domain ( an evolved o6-alkylguanine - dna alkyltransferase ) with the c - terminal 
 catalytic domain of a human adar enzyme ( adenosine deaminases acting 
 on rna ) . at the rna component , the incorporation 
 of a small chemical moiety , o6-benzylguanine 
 ( bg ) , is necessary . the covalent bond is then formed in situ in a 
 single - turnover enzymatic reaction between the snap - tag and the bg 
 moiety with very fast kinetics ( kconjugation = 2.8  10 m s ) and high specificity ( figure 1a ) . recently , we demonstrated 
 the repair of a premature stop codon ( uag ) into a tryptophan codon 
 ( uig ) in a fluorescent reporter gene in human cells ( 293 t ) . notably , the repair reaction was strongly dependent 
 on the covalent attachment of the guiderna to the deaminase . this 
 opens the appealing possibility of controlling the editing reaction 
 by triggering the assembly of the covalent rna we decided 
 to apply light as a trigger , as it allows for the very precise and 
 fast control in time , space , and dosage . to achieve the light - dependent 
 assembly of the covalent rna  protein 
 conjugate , we masked the bg moiety chemically by installment of a 
 light - sensitive 6-nitropiperonyloxymethyl ( npom ) protection 
 group which absorbs broadly in the 330420 
 nm range . during synthesis we obtained a separable 1:2 mixture of 
 regioisomers containing the npom group either at n7 or n9 position 
 of the guanine base . upon irradiation with 365 nm light on a common 
 uv - light table , both isomers , n7 and n9 , decay efficiently into free 
 bg and the respective nitroso acetophenone byproduct with similar 
 kinetics ( n7 isomer , figure 1b , t1/2 = 34  3 s ; n9 isomer , figure s13 , t1/2 = 
 47  4 s ) . the decaging efficiency  was determined 
 by comparison with a commercial standard ( dmnb - camp ) to be 2000 
 and 1500 m cm for 
 the n7 and n9 isomers , respectively , giving quantum yields  
  0.5 and 0.36 ( for details , see the supporting information ) . ( a ) concept of light - triggered site - directed rna editing . 
 assembly 
 of the guiderna  deaminase conjugate requires release of the 
 npom - protected benzylguanine ( bg ) moiety and is a prerequisite for 
 the editing reaction . ( b ) first - order kinetic analysis ( via hplc ) 
 of the photodeprotection of n7-bg at the small - molecule 
 level . the hplc trace shows the product mixture after 60 s of 365 
 nm irradiation ( 75% conversion ) . the respective analysis for n9-bg ( c ) light - triggered conjugation reaction of fluorescein - labeled 
 npom - bg with snap - adar1 protein ( sds - page coomassie versus fluorescein 
 stain ) . bg - fitc refers to the conjugate of bg with fluorescein isothiocyanate , 
 and bg - fam refers to the conjugate with 6-carboxyfluorescein . to determine the reactivity of 
 the npom - protected bg with snap - deaminases such probes were incubated with sub - stoichiometric amounts of 
 snap - adar1 either in the dark or in the presence of 365 nm light . the conjugate formation was then determined by sds - page and fluorescence 
 analysis ( figure 1c ) . it was clearly shown that full fluorescence labeling of the snap - deaminase 
 was readily accessible upon irradiation . however , some background 
 reactivity of the protected bg in particular of the n9 isomer with 
 snap - adar1 was visible . the latter is coherent with the requirement 
 of the natural ancestor of the snap - tag to accept the desoxyribose 
 at the n9 position of the nucleobase . we did not expect the low - level residual activity to play a role 
 under dilute conditions inside the living cell ; nevertheless , we continued 
 all further work with the pure n7 isomer of npom - bg . to study the assembly of the guiderna deaminase 
 conjugate and its effect on in vitro rna editing , we attached the 
 n7-bg via a short linker to the 5-terminal aminolinker of a 17 nt guiderna that directs 
 the conjugate to codon 66 of the ecfp transcript in order to stimulate 
 the repair of a premature amber stop codon ( uag ) back to tryptophan 
 ( figure 2a ) . via sds - page we first characterized the light - dependent assembly of the riboprotein 
 ( figure 2b ) . conjugation 
 results in a readily detectable shift of the snap - deaminase toward 
 higher molecular weight . indeed , excellent control of the conjugate 
 assembly was achieved in a clearly light - dose - dependent manner , shifting 
 the snap - deaminase from non - conjugated to nearly complete conversion 
 following kinetics agreeing with the decaging kinetics of the bg precursor described above . to study the light - dependent 
 in vitro rna editing reaction , a master mix containing all components 
 was aliquoted in the dark into pcr tubes and aliquots were irradiated 
 individually with 365 nm light for 0 , 15 , 30 , 60 , or 180 s , respectively , 
 prior to starting the editing reaction by incubation at 37 c . a guiderna lacking the bg moiety served as a negative , and a guiderna 
 modified with authentic bg served as a positive control . similarly , only very minor editing 
 above background was detectable in the non - irradiated sample with 
 the npom - caged bg - guiderna . however , upon irradiation editing was 
 restored in a light - dose - dependent manner regaining an editing level 
 comparable to that of the positive control ( figure 2c , 75% with snap - adar1 ; figure s16 , 60% with snap - adar2 ) . plotting the intermediate 
 editing levels against the irradiation time resulted in first - order 
 kinetics ( figure 2c , t1/2 = 26  0.5 s ) very similar to those 
 obtained with the small - molecule precursor ( figure 1b ) . ( a ) n7-bg is included into an activated linker that 
 readily reacts with the aminolinker of commercially available rna 
 analogues to obtain the bg - guidernas . ( b ) the light - driven 
 conjugation reaction between npom - bg - guiderna and snap - adar1 is easily 
 monitored by sds - page ( coomassie stain ) . ( c ) in vitro site - directed 
 rna editing of the amber stop codon at position 66 in the ecfp gene . the editing yield is clearly light - dose - dependent obeying first - order 
 kinetics . sanger sequencing of the entire orf of the ecfp gene shows 
 no off - target editing ( figure s15 ) . the 
 respective editing applying snap - adar2 instead of snap - adar1 is given 
 in figure s16 . for further details , see 
 the supporting information . for the study of intracellular light - activated rna 
 editing , we incorporated 
 the n7-bg into a 19 nt antagomir - like chemically stabilized nucleic acid analogue that contained a gap of three natural ribonucleotides 
 around the editing site . we applied antagomir - like chemistry to improve 
 the stability of the guiderna and the selectivity of the editing reaction ; this has been shown for rna interference before . the guiderna targets a premature amber stop 
 codon ( uag ) at an egfp reporter ( w58amber ) , and successful editing 
 is indicated by turn - on of egfp fluorescence . a guiderna with authentic 
 bg served as a positive and the same guiderna lacking the bg moiety 
 as a negative control . further controls were done to test the necessity 
 of all components of the machinery . snap - adar1 and the reporter gene 
 were transiently overexpressed from plasmids in hek293 t cells . one 
 day after transfection of the plasmids , the respective guiderna was 
 lipofected into the cells . four hours after lipofection , the medium 
 was changed and cells were irradiated with 365 nm light under high 
 control of dosage and wavelength by using a fluorescence microscope 
 equipped with a led light source . one day later , the egfp fluorescence 
 was analyzed by microscopy before the rna was isolated , and the editing 
 yield was determined by sanger sequencing . compared to our previous 
 protocol , we had to optimize the amounts and stoichiometry of snap - adar1 
 and guiderna in order to suppress some low - level ( 10% ) editing 
 caused by the npom - protected guiderna in the dark ( for details , see figures s19 and s20 ) . the optimal amount of guiderna 
 used was 10 pmol/150 l and is in a range typical for sirna 
 duplexes . applying the nh2-guiderna ( negative editing control ) , 
 only a very few cells developed a low - level gfp fluorescence and no 
 editing was detectable in the sequencing trace ( < 5% ) . however , 
 transfecting bg - guiderna ( positive editing control ) gave brightly 
 fluorescent cells , similar to the transfection of functional wt egfp , 
 and an editing yield of typically 45% was determined ( figures 3 and s17 ) . furthermore , absolutely no editing was obtained at codon 58 by a chemically 
 stabilized , negative control guiderna that directs repair to codon 
 66 . thus , the formation of the rna secondary structure directly at 
 the targeted codon is strictly required for site - directed rna editing 
 and is the major determinant of specificity . the npom - protected guiderna 
 gave only very low editing yield over background ( < 5% ) and only 
 a small number of low - intensity fluorescent cells . however , following 
 illumination , editing was switched on to a level similar to that of 
 the positive editing control , as indicated by fluorescence microscopy 
 but also by rna sequencing ( 45% editing yield ) . intermediate editing 
 levels have been accessible by varying the light dose ( figure 3 ; more details can be found 
 in figure s17 ) . the light dose applied 
 to photoactivate editing was well tolerated by the cells . in comparison 
 to the npom - guiderna , the editing yield of neither the positive nor 
 the negative editing controls was dependent on light ( figure s17 ) . no unspecific off - target editing 
 was observed in the reporter gene , as indicated by sanger sequencing 
 ( figure s18 ) . snap - adar1 and 
 the reporter gene ( w58x egfp , or wt egfp ) are provided on plasmids , 
 the guidernas are reverse - transfected , all as described in the supporting information . shown is the fluorescence 
 microscopy analysis together with the respective sanger sequencing 
 traces 24 h post - transfection of the respective guiderna . 5-terminal , 
 the guidernas are either carrying an aminolinker ( nh2 ) , 
 the bg moiety ( bg ) , or the npom - protected bg moiety . the stop66-guiderna 
 is a negative control bg - guiderna targeting the gfp gene around codon 
 66 instead of codon 58 . as it requires massive genetic manipulation to switch the 
 expression of one isoform to another that differs only in a single 
 point mutation , site - directed rna editing might offer a practical 
 alternative . a light - triggered variant 
 would be particularly attractive for developmental biology , as early 
 stages are often transparent , and the 
 spatiotemporal control of gene expression is of particular interest . an emerging model system for developmental and neurobiology is the 
 marine annelid platynereis dumerilii(29 ) that is readily cultivated and easily manipulated at the one - cell zygote by microinjection . to test site - directed rna editing inside the 
 worm , we injected two mrna transcripts encoding snap - adar1 and egfp 
 together with chemically stabilized 21 nt guidernas . one day after 
 microinjection , when the zygotes were developing into trochophore 
 larvae , the fluorescence phenotype was analyzed by microscopy ( figures 4 and s22 ) . a gfp - positive phenotype was only detectable 
 in the positive control ( wt gfp ) and in the editing sample ( figure 4a , f ) . all negative 
 controls lacking parts of the machinery , such as the guiderna , snap - adar1 , 
 or both , showed no green fluorescence ( figure 4b  d ) . in the editing sample as well 
 as in the positive control , there was some heterogeneity of fluorescence 
 intensity that may result from the difficulty of precisely controlling 
 the injection volume . to determine the editing yield , a cohort of 
 trochophores ( each 80100 ) were lysed , and rna was extracted 
 and analyzed by sanger sequencing ( figure 4 ; for detailed analysis of all larvae , see table s23 ) . editing was observed only when all 
 components were included and achieved 6070% over the entire 
 population . no off - target editing was observed in the targeted transcript 
 ( figure s24 ) . the worms seem to develop 
 and behave normally over the first days and stages of development . reporter 
 mrna ( egfp ) and snap - adar1 mrna were microinjected into one - cell zygotes , 
 together with the respective bg / nh2-guiderna and rhodamine are the fluorescence images of one 
 representative embryo 24 hours post fertilization ( hpf ) for each experiment 
 and the sequencing trace obtained from the rna of 80100 animals 
 per experiment : ( a ) positive control , ( b  e ) negative controls 
 lacking single components of the editing machinery , and ( f ) editing 
 experiment . for details , see the supporting information . deaminase 
 conjugate and fails when using the nh2-guiderna lacking 
 the bg moiety ( figure 4e ) . in contrast to using the nh2-guiderna ( figure 4e ) lacking the bg 
 moiety , microinjection of the npom - protected guiderna resulted in 
 a small but significant number of faintly fluorescing trochophores 
 ( 18% ) besides a large number of dark ones ( > 80% , figure 5a , table s23 ) . in accordance with this , rna sequencing of a cohort of 
 80100 animals revealed a low but significant residual editing 
 at the targeted stop codon ( 10% ) . in faintly fluorescent trochophore larvae , the rhodamine signal was typically stronger ( figure 5a ) , indicating that the low - level 
 editing might be due to an undesirably high injection volume of the 
 editing components . this low - level residual editing activity is reminiscent 
 of the situation described above for the light - dependent editing in 
 cell culture . however , when the microinjected trochophores were treated 
 with 365 nm light on a uv trans - illuminator ( 5 min ) , half of the trochophores 
 developed a bright egfp signal ( figure 5b , table s23 ) . the fluorescence 
 imaging was in agreement with an editing yield of 60% , as 
 determined by rna sequencing of 80100 animals ( figure 5b ) . thus , irradiation allows 
 for activating rna editing to a yield nearly identical to that of 
 the positive editing control with an unprotected bg moiety ( figure 4f ) . reporter 
 gene and snap - adar1 have been microinjected into one - cell platynereis zygotes as described in figure 4 , but now with a photoprotected chemically 
 stabilized bg - guiderna . within 1 h after microinjection , zygotes have been ( a ) kept in the dark or ( b ) treated with 365 nm 
 light ( 5 min ) . fluorescence images and rna sequences ( 80100 
 animals / experiment ) are taken 24 hpf . the sequence of the guiderna 
 is the same as in figure 4 . for further details , rna - guided 
 enzymes represent rationally programmable tools that 
 allow for the efficient and precise manipulation of nucleic acids 
 at specific sites in living organisms . here , we further elaborate 
 a novel strategy for site - directed adenosine - to - inosine rna editing 
 ( a ) by introducing photocontrol and ( b ) by applying the tool in developing platynereis dumerilii . the presented approach is unique 
 in that the artificial rna - guided 
 editing enzyme is assembled via the formation of a single covalent 
 bond . as covalent bond formation is essential 
 for the functioning , photocontrol is feasible by blocking the snap - tag - mediated 
 bond formation via installment of a single photoprotection group at 
 the o6-benzylguanine moiety . specifically , 
 we demonstrate the ready synthesis of npom - protected bg and its convenient 
 introduction into diversely chemically modified antisense oligomers 
 after their solid - phase synthesis . this is in contrast to other strategies 
 that require the site - specific incorporation of ( often several ) photoprotected 
 nucleosides during solid - phase oligonucleotide synthesis to achieve 
 photocontrol of biochemical processes . furthermore , our strategy needs less genetic engineering 
 compared to the introduction of photoresponsive groups into enzymes 
 by means of amber suppression or related strategies . in vitro we could show that the attachment of the npom group 
 at 
 n7 , but not n9 , of the bg moiety blocks the conjugation reaction with 
 the snap - tag . however , reactive benzyl guanine is readily released 
 upon 365 nm irradiation with high efficiency (   
 2000 m cm ) and allows for 
 the light - dose - dependent assembly of guiderna  deaminase conjugates . besides editing , the npom - protected bg will be applicable in other 
 approaches that rely on the snap - tag , like chemical inducers of dimerization . by controlling the assembly of the editase , 
 we could trigger the in vitro editing of a purified mrna in a light - dose - dependent 
 manner covering the whole dynamic range from absence of editing in 
 the absence of light until full editing in the presence of light . the desired action of our tool could be directly translated into mammalian 
 cell culture ; however , optimization was required to control low - level 
 residual editing by the photoprotected guiderna . furthermore , we established site - directed 
 rna editing for the first time in a living organism . specifically , 
 we achieved the efficient and highly selective switch of a premature 
 stop into a tryptophan codon in developing platynereis dumerilii zygotes . notably , no genetic engineering and livestock breeding 
 is required , thus circumventing time - consuming and cost - intensive 
 laboratory work . as our editing tool is independent of any host - specific 
 factors , the technology should be transferable to any other organism . 
 in platynereis , the covalent assembly of the guiderna deaminase 
 conjugate was again essential , and our simple photocontrol strategy 
 for site - directed rna editing was directly transferable . the tool 
 could now be further elaborated to achieve precise spatiotemporal 
 control of protein isoforms in cellular networks or in developing platynereis . trifluoroacetamide 
 protected o6-benzylguanine 
 ( bg , 120 mg , 0.33 mmol ) was solved in 
 dry dmf ( 1.2 ml ) under argon . diazabicycloundecene ( 150 
 l , 153 mg ) was added at room temperature , and the solution 
 was stirred for 30 min . npom chloride ( 0.5 mmol , 1.5 equiv , 
 dissolved in 1.6 ml of dmf ) prepared in situ as described was added dropwise . after 2.5 h , the reaction 
 mixture was diluted with etoac , washed with 1% citric acid ( 3 ) 
 and brine ( 1 ) , and dried over na2so4 . the evaporated crude product was cleaned via silica chromatography 
 ( 24% meoh in dcm ) and yielded 24 mg ( 21% ) of npom - bg - tfa and 50 mg ( 42% ) of npom - bg - tfa . for full 
 characterization and assignment of the isomers and downstream synthesis , h nmr ( 600 mhz , dmso - d6 ) :  = 9.99 ( t , j =   5.9 hz , 1h ) , 8.07 ( s , 
 1h ) , 7.48 ( d , j =   8.1 hz , 
 2h ) , 7.35 ( s , 1h ) , 7.28 ( d , j =   8.1 hz , 2h ) , 6.84 
 ( s , 1h ) , 6.22 ( s , 2h ) , 6.16 ( s , 1h ) , 6.04 ( s , 1h ) , 5.54 ( m , 2h ) , 5.46 
 ( m , 2h ) , 5.13 ( q , j =   6.2 hz , 1h ) , 4.39 ( d , j =   5.9 hz , 2h ) , 1.33 ( d , j =   6.2 hz , 
 3h ) . c nmr ( 151 mhz , dmso - d6 ) :  = 164.1 , 159.8 , 156.4 ( q , j(c , f ) = 36 hz ) , 156.3 , 151.8 , 146.5 , 145.9 , 140.5 , 137.2 , 136.2 , 
 135.5 , 127.9 , 127.5 , 116.0 ( q , j(c , f ) 
 = 288 hz ) , 105.6 , 105.2 , 104.3 , 103.2 , 74.9 , 72.0 , 66.6 , 42.4 , 23.3 . hr - esi - ms : [ m + h](theoretical ) = 590.16056 
 for c25h23f3n7o7 ; found 590.16118 . h nmr ( 600 mhz , dmso - d6 ) :  = 10.00 ( t , j = 
 6.0 hz , 1h ) , 7.80 ( s , 1h ) , 7.49 ( d , j = 8.1 hz , 2h ) , 
 7.46 ( s , 1h ) , 7.31 ( d , j = 8.1 hz , 2h ) , 6.97 ( s , 
 1h ) , 6.34 ( s , 2h ) , 6.15 ( s , 1h ) , 6.03 ( s , 1h ) , 5.435.49 ( m , 
 2h ) , 5.40 ( d , j = 11.4 hz ) , 5.32 ( d , j = 11.4 hz ) , 5.21 ( q , j = 6.3 hz , 1h ) , 4.41 ( d , j = 6.0 hz , 2h ) , 1.38 ( d , j = 6.3 hz , 3h ) . c nmr ( 151 mhz , dmso - d6 ) :  = 160.8 , 157.2 ( q , j(c , f ) = 36 hz ) , 155.3 , 152.7 , 147.5 , 142.1 , 140.5 , 138.2 , 
 137.0 , 136.6 , 129.6 , 128.3 , 116.9 ( q , j(c , f ) = 288 hz ) , 114.3 , 106.7 , 105.2 , 104.1 , 73.1 , 71.7 , 67.5 , 43.3 , 
 24.1 . hr - esi - ms : [ m + na](theoretical ) = 612.14250 
 for c25h22f3n7o7na ; found 612.14262 . purified snap - adar1 
 ( 170 nm ) , purified ecfp mrna ( 10 nm ) , and one of the respective guidernas 
 ( 50 nm ) were prepared in buffer ( 25 mm trishcl , 0.75 mm mgcl2 , 75 mm kcl , 2 m heparin , and 640 u / ml murine rnase 
 inhibitor , 10 mm dtt , ph 8.3 ) in pcr tubes . irradiation with 365 nm 
 light was performed on a uv trans - illuminator ( uvp tfl-40v , 25 w , 
 intensity high ) for the indicated amount of time at room temperature . subsequent editing was performed by incubation for 120 min while cycling 
 between 30 and 37 c . reactions were stopped by heating to 70 
 c for 3 min and subsequent reverse transcription . after pcr 
 amplification of the cdna , editing yields were estimated by the relative 
 height of the guanosine versus adenosine traces by sanger sequencing . sequence of 
 the guidernas : ( npom)bg / nh2-ucg - gaacaccccagcacaga-3 
 ( natural ribonucleotides ; 5-terminal modifications were introduced 
 via amino - linker , the 5-terminal three nucleotides serve as 
 linker and do not base - pair with the target ) . cells ( 293 t : dsmz 
 code acc-635 ; 200  000 cells / well ) were seeded on 24-well plates 
 in full media ( dmem , 10% fbs , 1% penicillin / streptomycin , grown in 
 5% co2 , 37 c ) . at 6080% confluency , plasmid 
 pcdna3.1 vector ( life technologies ) carrying snap - adar1 ( 100 ng / well ) 
 and pcdna3.1 vector carrying the respective egfp variant ( 500 ng / well ) were co - transfected with lipofectamine 2000 
 ( 4 l/g ) . after 24 h , the 
 cells were reverse transfected into 96-well plates ( 60 000 cells / well ) 
 containing the respective guidernas ( 10 pmol / well ) pretreated with 
 lipofectamine 2000 ( 0.5 l / well ) . four hours after reverse transfection , 
 media was replaced with dmem without fbs and phenol red , containing 
 hepes ( 25 mm ) . irradiation ( 365 nm ) was performed in a fluorescence 
 microscope ( zeiss cellobserverz.1 , equipped with a 365 nm colibri.2 
 led ) at 100% led power for the indicated amount of time . twenty - four 
 hours later , the fluorescence phenotype was analyzed by fluorescence 
 microscopy ( zeiss cellobserverz.1 ) , and rna was extracted using the 
 rneasy minelute cleanup kit ( qiagen ) . after reverse transcription 
 and pcr amplification , the editing yield was estimated by sanger sequencing . the sequence 
 of the w58x guidernas was ( npom)bg / nh2-usasu - gugucgg - cca - cggaascsasgsg-3 ; 
 the sequence of the stop66-guiderna was bg - uscsg - gaacacc - cca - gcascsasgsa-3 ( s = phosphothioate 
 linkage ; plain font indicates 2-methoxyribonucleotides , and 
 italic underlined indicates unmodified ribonucleotides ; the three 
 5-terminal nucleotides serve as a linker and do not base - pair 
 with the target ) . for 
 the in vitro transcription of stabilized mrnas of snap - adar1 and egfp 
 variants with the mmessage mmachine t7 ultra kit ( life technologies ) , 
 the respective genes were subcloned into the puc57-t7-rpp2 vector , 
 resulting in 5-capped and 3-polyadenylated transcripts 
 additionally stabilized by a platynereis - specific 
 rpp2 5-utr , as described before . fertilized zygotes were obtained from an in - house breeding 
 culture and were incubated at 14.8 c for 55 min . prior to microinjection , 
 the egg jelly was removed by rinsing the zygotes with natural seawater 
 ( nsw ) in a 100 m sieve . to soften the vitellin envelope , a 
 1-min - long proteinase k treatment ( 70 g / ml ) 100 zygotes 
 were embedded in the injection stage ( 2% agarose in nsw ) . samples 
 were injected using femtotipps ii microcapillaries with a femtojet 
 express microinjector ( 700 hpa injection pressure , 0.1 s injection 
 time , 35 hpa compensation pressure ) in a cooled ( 14.8 c ) zeiss 
 axiovert 40 cl microscope equipped with a luigs and neumann micromanipulator 
 as described before . injection started 
 1 hours post fertilization ( hpf ) and was stopped when the first cleavage 
 was detected ( ca . irradiation at 365 nm was performed immediately 
 after microinjection for the indicated amount of time on a uv trans - illuminator 
 ( uvp tfl-40v , 25 w , intensity high ) . microinjected zygotes were bred 
 at 19 c in nunclon six - well plates containing 6 ml nsw . twenty - four 
 hpf , healthy larvae ( early trochophore ) were separated from unhealthy 
 ones . rna from 80100 healthy larvae ( two injection sessions ) 
 was isolated 25 hpf by shock freezing ( liquid nitrogen ) , shear forces 
 ( passing through 0.6 mm needle ) , vortexing ( 10 s ) , and subsequent 
 use of the rneasy minelute cleanup kit ( qiagen ) . after reverse transcription 
 and pcr amplification , the editing yield was determined by sanger 
 sequencing . injection samples contained 1.5 g/l rhodamine - dextran 
 ( 10 kda mw , sigma ) for injection control , 250 ng/l of the respective 
 reporter mrna , 450 ng/l snap - adar1 mrna , and 25 m of 
 the respective guiderna . sequence of the guiderna : bg / nh2-usasu - gugucgg - cca - cggaacasgsgscsa-3 
 ( s = phosphothioate linkage ; plain font indicates 2-methoxy 
 ribonucleotides , and italic underlined indicates unmodified ribonucleotides ; 
 the 5-terminal three nucleotides serve as linker and do not 
 base - pair with the target ) .