TargetingN-TerminalHuntingtinwithaDual-sgRNA ...downloads.hindawi.com/journals/bmri/2019/1039623.pdfincluding caspases, calpains, cathepsins, matrix metal-loproteinases,andasparticproteases,thatcleaveHTThave

Research ArticleTargeting N-Terminal Huntingtin with a Dual-sgRNAStrategy by CRISPRCas9

Junjiao Wu12 Yu Tang 1 and Chun-Li Zhang 34

1National Clinical Research Center for Geriatric Disorders Department of Geriatrics Xiangya HospitalCentral South University Changsha Hunan Province China2Department of Rheumatology and Immunology Xiangya Hospital Central South University Changsha Hunan Province China3Department of Molecular Biology University of Texas Southwestern Medical Center Dallas TX USA4Hamon Center for Regenerative Science and Medicine University of Texas Southwestern Medical Center Dallas TX USA

Correspondence should be addressed to Yu Tang tangyu-sky163com and Chun-Li Zhang chun-lizhangutsouthwesternedu

Received 30 April 2019 Revised 30 July 2019 Accepted 5 September 2019 Published 16 November 2019

Academic Editor Marco Fichera

Copyright copy 2019 Junjiao Wu et al 2is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Huntingtonrsquos disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by a CAGpolyglutamine(polyQ) repeat expansion in the Huntingtin (HTT) gene 2e polyQ tract is located in and transcribed from N-terminal HTT ofexon 1 HTT is a large multifaceted protein which participates in a range of cellular functions Previous studies have shown thattruncated HTT which lacks N-terminus retains specific functions that can produce neuroprotective benefits It gives an insightthat it is possible to repair HD by removing deleterious N-terminal HTTwith CRISPRCas9 without compromising functions ofremaining HTTpeptides To successfully generate functional truncated HTTproteins an alternative downstreamATG start codonthat is capable of initiating truncated HTT expression is required In this study we searched all possible in-frame ATGs beforeexon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids We then tried toremove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPRCas9 however we cannot detectobvious traits of truncated HTTexpression Our results suggest that noncanonical ATGs of N-terminal HTTmay not be effectivein the genomic context as in the construct context Nevertheless our study examined the therapeutic efficacy of downstreamnoncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manip-ulation of the HTT gene

1 Introduction

Huntingtonrsquos disease (HD) is an autosomal dominantneurodegenerative disease caused by the expanded CAGtract resided in the first exon of the Huntingtin (HTT) gene[1 2] Basically the pathogenic mutant HTTconsists of morethan 35 CAG repeats which are then translated into pol-yglutamine (polyQ) proteins [3 4] Although HTT isubiquitously expressed in the body expanded polyQ pro-teins may form intracellular aggregates and preferentiallycause the loss of medium spiny neurons (MSNs) in thestriatum via a gain of toxic function [5] 2e CAG tractlength determines HTT propensity for aggregation and

toxicity and is inversely correlated with age of onset in HDpatients [6]

HTT is a multiple conformation protein of normally3144 amino acids and has conserved N-terminal sequences[7 8] By binding with other proteins HTT is considered tobe multifaceted that is essential for a spectrum of cellularfunctions such as embryonic development antiapoptoticpathway BDNF modulation ciliogenesis autophagy andvesicular transport [7] N-terminal HTT containing the first17 amino acids (N17) and the polyQ tract can be formed byproteolytic cleavage and is one of the most widely studiedHTT peptides HTT proteolysis in patient brains is thecascade of various cleavage events Numerous proteases

HindawiBioMed Research InternationalVolume 2019 Article ID 1039623 10 pageshttpsdoiorg10115520191039623

including caspases calpains cathepsins matrix metal-loproteinases and aspartic proteases that cleave HTT havebeen reported [9ndash13]

2e N17 domain as an evolutionarily conserved domainamong vertebrates functions as a nuclear export signal(NES) [14] and is necessary for nuclear exclusion of smallmutant HTT fragments thereby modifying nuclear patho-genesis and disease severity [15] 2e N17 domain forms anamphipathic α-helical structure that signals endoplasmicreticulum (ER) stress [16] and also senses reactive oxygenspecies (ROS) stress specifically through direct oxidation ofa highly conserved methionine at position 8 [17] Moreoverthe polyQ tract can act as a flexible domain allowing theflanking domains to come into close spatial proximity toform proper functional conformations [18] however thisflexibility may be impaired with expanded polyQ tractsImportantly multiple posttranslational modifications suchas phosphorylation ubiquitination acetylation andsumoylation have been identified in the N17 domain thatmay affect the clearance of HTT and its subcellular locali-zation [19ndash21] 2us N-terminal HTT is considered to beimportant in regulating protein aggregation subcellularlocalization and cytotoxicity Despite of this fact othermodification sites in the remaining HTT protein have beendemonstrated to possess pivotal cellular functions as well[7] For instance sequential N-terminal proteolysis of HTTalso generates a C-terminal fragment which induces cyto-toxicity via abnormal vacuolation of ER and increases ERstress [22] Interestingly without a whole stretch ofN-terminus however HTTmay also retain some beneficialfunctions [23 24] For example Liu et al reported that atruncated HTT lacking the first 237 amino acids spanningexon 1sim6 can rescue the HTT deficiency-induced neuriticdegeneration which largely depends on the dynein-bindingregion known to be involved in intracellular trafficking [24]

So far different strategies have been employed to pro-duce therapeutic effects in both cellular and animal modelsof HD including traditional avenues such as RNA in-terference (RNAi) [25ndash27] and antisense oligonucleotides(ASOs) [28ndash31] pharmacological treatments [32 33] andthe emerging genome editing by CRISPRCas9 both in vitroand in vivo [34ndash39] A recent study by Southwell et aldeveloped ASOs targeting HD single-nucleotide poly-morphisms (SNPs) that can selectively suppress mutantHTT expression in the mouse HD model [31] 2ey alsoachieved preclinical efficacy data of those HTT ASOs de-livered to cynomolgus monkeys via lumbar puncture [31]Similarly two other recent studies have used an allele-se-lective CRISPRCas9 strategy based on protospacer adjacentmotif (PAM) sites created by altering SNPs thereby selec-tively inactivating mutant HTT allele for a given diplotype[34 35] As this is a quite novel personalized strategy itrequires a comprehensive analysis of patientsrsquo HTT hap-lotype patterns 2e nonallele-selective inactivation of HTThas also been achieved by using a pair of sgRNAs flankingCAG repeats in a transgenic HD mouse model [36] as wellas in patient-derived fibroblasts with the help of Cas9nickase [37] CRISPRCas9 was also employed to correct theHTT mutation in an HD-induced pluripotent stem cell

(iPSC) line which can rescue the HD phenotypes such asimpaired neural rosette formation increased susceptibilityto growth factor withdrawal and deficits in mitochondrialrespiration [38] Notably CRISPRCas9 can permanentlydisrupt the targeted genes rendering it to be a more efficienttherapeutic approach than those with continuous delivery ofdrugs for treatment However it also brings about the ca-veats that the treatment cannot be reversed in the event ofadverse effects

Taking advantage of the higher efficiency by the CRISPRCas9 system we hypothesize that we may remove a length ofN-terminal HTT to abolish the neurotoxicity stressed bypolyQ aggregations and meanwhile keep the functions ofprobably generated truncated HTT after CRISPR targetingTo successfully obtain this type of endogenous truncatedHTT it is required that an alternative downstream ATGstart codon will be capable of initiating truncated HTTtranslation albeit at lower levels 2erefore in this study wetried to search all possible in-frame ATGs within the first 6exons that coded for 249 amino acids which were thencloned to fuse with GFP in plasmids and we demonstratedthat one of them can indeed start downstream translationHowever after removing endogenous N-terminal HTT byan optimized dual-sgRNA targeting we failed to detectobvious traits of truncated HTT expression Our resultssuggest that noncanonical ATGs of N-terminal HTT maynot be effective in the genomic context as in the constructcontext

2 Materials and Methods

21 Cloning and Sequencing Constructs of E3a- E3b- E4-and E6-GFP were generated based on the pcDNA31(+)vector using Gibson Assembly Master Mix (New EnglandBiolabs MA USA) Specifically different lengths of HTTfragments were amplified from a human cDNA library with21 CAGs located in N-terminus E3a∆- E3b∆- E4∆- andE6∆-GFP plasmids were then respectively constructed byPCR using primers flanking exon 2 and 5primeUTR from theircorresponding plasmids and finally self-ligated into circleplasmids 2e length of 5primeUTR in all constructs is 145 bpwhich is exactly the full-length of 5primeUTR in the NM_002111transcript All pcDNA constructs were finally confirmed bySanger sequencing For genome editing lentiCRISPRv2(Addgene 52961) was used as the backbone and sgHTTsequences were designed with the help of CHOPCHOPwebsite (httpchopchopcbuuibno) In brief sgRNAoligos were annealed and inserted into the BsmBI digestedvector Junctions of exon 12 or targeted genomic regionswere amplified by PCR and ligated into the pMD20T vector(Takara Bio Inc CA USA) for T-A cloning White colonieswere picked out for plasmid extraction and Sanger se-quencing Sequences of sgHTTand PCR primers are listed inTable 1

22 Cell Culture HEK293 cells were maintained in theDMEM medium (2ermo Fisher Scientific MA USA)supplemented with 10 fetal bovine serum (Corning NY

2 BioMed Research International

USA) and 1 penicillinstreptomycin at 37degC and 5 CO2For transfection passage and seed HEK293 cells at a densityof 2times105 cells per well of a 6-well plate 2e same amount of05 μg constructed pcDNA plasmids were then transfectedby the polyethylenimine (PEI) reagent GFP signals andprotein aggregations were observed 24 hrs later For CRISPRtargeting HEK293 cells were transfected with differentsgRNA pairs (05 μg05 μg mixture) by the PEI reagentAfter 48 hrs collect HEK293 cells for indel analysis by theT7E1 assay For clonal cell line selection passage targetedcells with 025 trypsin and further dilute and seed 6000single cells in a 10 cm dish Single colonies were picked outunder a microscopy and further examined by junction PCRPCR primers are listed in Table 1

23 T7E1 Assay 2e cleavage efficiency of dual sgHTT wasexamined by the T7E1 assay accordingly to the previouslyreported method [40] In brief transfected cells weredigested and spun down at 250 g for 4min and later lysedwith DNA lysis buffer (100mM Tris-HCl (pH 80) 25mMEDTA (pH 80) 100mMNaCl 1 Triton X-100 and 50 ngμl RNase A) containing 05mgml Proteinase K at 55degC forovernight DNA lysates were used directly as templates forgenomic PCR T7E1 primers are listed in Table 1 PCRproducts were then melted and reannealed to form het-eroduplexes which were then digested by T7E1 (New En-gland Biolabs) and electrophoresed in 2 agarose gelsCalculate the fraction of the PCR product cleaved (fcut) byusing the following formula fcut (b+ c)(a+ b+ c) where ais the integrated intensity of the undigested PCR productand b and c are the integrated intensities of each cleavageproduct Indel occurrence was estimated with the followingformula indel () 100times (minus

(1 minus fcut)

1113968)

24 Gene Expression Gene expression was performed ac-cordingly to the previously reported method [41] TotalmRNAs were isolated from cultured cells using TRIzol(Invitrogen Carlsbad CA USA) and were reverse tran-scribed into cDNAs using a SuperScript III kit (Invitrogen)Quantitative PCR was then performed using SYBR GreenERSuperMix (Invitrogen) on an ABI 7900HT thermocycler(Applied Biosystems) 2e PCR program consisted of 2minat 50degC and 10min at 95degC followed by 40 cycles of 15 sec at95degC and 45 sec at 62degC Relative expression levels wereanalyzed using the 2minus ΔΔCt algorithm normalized to thehousekeeping gene GAPDH Primer sequences are listed inTable 1

25 Western Blot Total proteins were extracted from cul-tured cells and homogenized mice brain tissues respectivelywith RIPA lysis buffer Proteins were then separated by SDS-PAGE with Tris-glycine gels (8 for HTT 15 for GFP) andtransferred onto PVDF membranes (Millipore MA USA)Nonspecific sites were blocked in 5 nonfat milk for 1 hr atroom temperature 2e blots were then probed at 4degC forovernight with primary antibodies against the followingproteins HTT (1 1 000 D7F7 5656 cell signaling) GFP(1 5 000 GFP-1020 Aves Labs) and α-tubulin (1 10 000T5168 Sigma) After washing with PBS for 3 times sec-ondary antibodies conjugated with horseradish peroxidase(1 25 000) from Jackson ImmunoResearch (711-035-152715-035-150 and 703-035-155) were incubated respectivelywith blots for 2 hrs at room temperature Protein bands werefinally visualized with the Pierce ECL substrate (2ermoFisher Scientific)

26 Statistical Analysis Results were presented asmeansplusmn SD values Statistical significance (plt 005) wasdetermined using Studentrsquos t-test

3 Results and Discussion

31 Capability of Initiating Translation by NoncanonicalATGs Functional truncated HTT is lack of N-terminal 237amino acids which are translated from within the first 6exons [24] To figure out any possible alternative ATG startcodon that may initiate its downstream protein translationand produce functional truncated HTT we first searched allin-frame ATGs before exon 7 We then identified fourcandidate ATGs among which two are located on exon 3(E3a-ATG and E3b-ATG) one on exon 4 (E4-ATG) andone on exon 6 (E6-ATG) (Figure 1(a)) Interestingly thesecandidate ATGs mostly conform to the consensus Kozakrule NNN(AG)NNATGG for eukaryotic cells suggestive ofstrong initiation potentials (Figure 1(a))

To test which of these ATGs can indeed start down-stream protein translation we then constructed differentlengths of HTT fragments harboring candidate ATGs re-spectively and fused them with GFP in plasmids(Figure 1(b)) Constructs of E3a- E3b- E4- and E6-GFPincluded exon 1 and thus preferentially used E1-ATG to starttheir translation However exon 1 and E1-ATG were all

Table 1 Sequences of sgRNAs and PCR primers

Sequences (5prime-3prime)sgRNA sequencessgHTT-1 GAGTCGGCCCGAGGCCTCCGsgHTT-2 GCCTCCGGGGACTGCCGTGCCsgHTT-3 GCGGGGACTGCCGTGCCGGGCsgHTT-4 GTGCCGGGCGGGAGACCGCCAsgHTT-5 GTGAGGAAGCTGAGGAGGCGGsgHTT-6 GGCTGAGGAAGCTGAGGAGGsgHTT-7 GGCGGCGGCTGAGGAAGCTGsgHTT-8 GAGCAGCGGCTGTGCCTGCGGsgHTT-9 GAGCGGCTCCTCAGCCACAGCT7E1 assay and junctionPCRHTT-T7E1-F CCAGCCATTGGCAGAGTCCGHTT-T7E1-R TTGCTGGGTCACTCTGTCTCTGQuantitative PCRHTT-qF TGACGCAGAGTCAGATGTCAGHTT-qR CCGAGGGGCACCATTCTTTTGAPDH-qF CGGGATTGTCTGCCCTAATTATGAPDH-qR GCACGGAAGGTCACGATGTSequencing of junctionsHTT-5primeUTRe1-F CGGGAGACCGGTGGCTGAGGHTT-e4e5-R CCGAGGGGCACCATTCTTTT

BioMed Research International 3

ACCGCCAUGG

AUCGCUAUGG

E1 ATG

E3a ATG

E3b ATG

E4 ATG

E6 ATG

GCUUUGAUGG

GUCAGGAUGG

AAAAUUAUGG

(a)

E6-GFPE4-GFP

E3b-GFPE3a-GFP

E6∆-GFPE4∆-GFP

E3b∆-GFPE3a∆-GFP 5rsquoUTR

5rsquoUTR5rsquoUTR5rsquoUTR


E2E2E2

E2E2E2E2

E3

E3E3E3E3

E3E3E3

E4E4

E4E4

E5

E5

E6

E6EGFPEGFPEGFP

EGFPEGFPEGFPEGFP

E1-ATGE1-ATGE1-ATGE1-ATG

PolyQ

5rsquoUTR EGFP

E2

(b)GFP

E6-G

FPE4

-GFP

E3b-

GFP

E3a-

GFP

610 620 630

530 540

710 720

930 940

T C A G AT G T C A G G G T G A G C AA G G GC

A A A G T T A T CA A A G T G A G C A A G G GC

G A A AT T A A A A A G G T G A GC AA G GG C

AA T G A A A T T AA G G T G A GC AA G G GC

(c)

E6∆-

GFP

E4∆-

GFP

E3b∆

-GFP

E3a∆

-GFP

Exon 25rsquoUTR

180 190 200

180 190 200

180 190 200

180 190 200

C G G G A G AC C G C C A A A G A A A G A AC T

C G G G AG AC C G C C AA A G A A A GA AC T

C G G G AG AC C G C C A A AG A A AG A AC T

C G G G AG AC C G C C A A A G A A AG A AC T

(d)

GFP

E3a-GFP E3a∆-GFP

E3b-GFP E3b∆-GFP

E4-GFP E4∆-GFP

E6-GFP E6∆-GFP

(e)

E6Δ-GFPE4Δ-GFP

E3bΔ-GFP

E3aΔ-GFP

GFP

α-Tubulin

GFP

43kDa

34kDa

E6Δ-GFP

E4Δ-GFP

E3bΔ

-GFP

E3aΔ

-GFP

26kDa

268kDa286kDa

300kDa

324kDa405kDa

GFP

lowast

lowastlowast

lowast

(f )

Figure 1 Capability of initiating translation by noncanonical in-frame ATGs (a) Candidate in-frame ATGs before exon 7 of HTT(b) Constructs of HTTfragments fused with GFP (c d) Sanger sequencing results of junctions in plasmid constructs (e) Transfection ofconstructs in HEK293 cells Scale bar 100 μm (f ) Western blot analysis of fused proteins Asterisks represented the E3a-ATG initiatedtranslation products


deleted in E3a∆- E3b∆- E4∆- and E6∆-GFP constructs(Figure 1(b)) All constructs retained the 5prime untranslatedregion (5primeUTR) of HTTtomaintain the upstream regulatorymachinery Sanger sequencing of those constructs con-firmed that ATG codons of GFP were all removed(Figure 1(c)) and that 5primeUTR and exon 2 were seamlesslyligated in truncated constructs (Figure 1(d))

After transfected into HEK293 cells with the sameamount constructs containing exon 1 did produce strongGFP signals and also led to extensive polyQ aggregations inthe cytoplasm (Figure 1(e)) 2is is in line with previousstudies that the NES located on the N17 region of exon 1exported HTT from the nucleus to cytoplasm [14] In-terestingly all of the truncated constructs (E3a∆- E3b∆-E4∆- and E6∆-GFP) can produce GFP signals evenly in thenucleus and cytoplasm as those in the GFP-alone controlgroup (Figure 1(e)) Furthermore western blot analysisshowed that different lengths of HTT fragments were suc-cessfully translated and fused with downstream GFP al-though the productivities of E4∆-GFP and E6∆-GFP weregreatly decreased (Figure 1(f)) Based on the sizes of proteinbands (286 kDa for E3a∆-GFP 300 kDa for E3b∆-GFP324 kDa for E4∆-GFP and 405 kDa for E6∆-GFP) wedemonstrated that E3a-ATG is responsible for the trans-lation of those fused proteins (Figure 1(f ) and Figure 2) 2eunspecific bands were probably derived from proteolyticcleavage of translated peptides Taken together these resultssuggest that alternative in-frame ATGs can initiate down-stream GFP expression in plasmids

32 Targeting N-Terminal HTTwith a Dual-sgRNA StrategyN-Terminal HTT is transcribed from exon 1 just after5primeUTR containing the conserved N17 region polyQ andpolyproline (polyP) tract In light of the initiation capabilityof downstream ATGs in plasmids we proposed to endog-enously delete the polyQ tract from N-terminal HTT byCRISPRCas9 in hope of maintaining the functions ofremaining HTT peptides

First we designed a range of sgRNAs flanking thepolyQ region with 4 upstream sgRNAs and 5 downstreamsgRNAs (Figure 3(a) and Table 1) We then used differentcombinations of sgRNA pairs to transfect cells and laterexamine their combined cleavage efficiencies by the T7E1assay Although this analysis would be a mixture of in-dependent cleavage events induced by each single sgRNAand deletion events induced by the dual sgRNAs we cannevertheless figure out the sgRNA pairs with highercleavage efficiencies Of those upstream sgHTT-1 andsgHTT-4 combined with downstream sgRNAs were shownto be highly efficient in cutting genomic DNA than thosewith sgHTT-2 and sgHTT-3 (Figure 3(b)) To retain 5primeUTRregulatory elements at the most and avoid the possibleregulatory interference by uncut polyP sequences we thuschose sgHTT-49 as the optimized sgRNA pair We thentransfected HEK293 cells and later screened out 12 singlecolonies We did junction PCR and finally selected one cellcolony (sgHTT-49 7) which is shown to be homozygousfor further analysis (Figure 3(c)) We then sequenced 7

gRNA and found that polyQ was seamlessly deleted fromN-terminal HTT (Figure 3(d))

Since the downstream sgHTT target sites are adjacent tothe exon 1intron 1 boundary this may disrupt the exonintron junction by indels and then affect the RNA splicingprocess A previous study has demonstrated that targetingthe junction of HTTexon 1intron 1 with CRISPRCas9 canreduce half of the mRNA level in the YAC128 mouse model[39] We thus harvested mRNAs from cell colonies andreverse-transcribed them into cDNAs for integrity analysisWe sequenced 7 cDNA and found that the junction of exon1exon 2 was still intact suggesting that no indels occurredin this region (Figure 3(d)) We next did quantitative PCRand the results showed a sim23 reduction of the HTT level incolony 7 and a sim54 reduction in colony 10 (Figure 3(e))2is result echoes the heterozygous property of colony 10but meanwhile documents that dual-sgHTT targeting inthis colony may destroy HTT transcripts by certain mech-anisms such as nonsense-mediated decay (NMD) that willeliminate mRNAs containing premature stop codons [42]Moreover sgHTT-9 targeting may have produced indels todisrupt the exon 1intron 1 junction in colony 10 finallydampening the transcription

We hence extracted proteins and performed western blotto examine the HTT expression We can observe full-lengthHTT band and also the cleaved fragments in WT HEK293cells (Figure 3(f)) Unexpectedly in the sgHTT-49-7 cellcolony there were no obviously visible HTT bands includingany possible truncated HTT bands suggesting the ineffectiveor extremely weak initiation capability of E3a-ATG in thisendogenous condition (Figure 3(f )) Note that the primaryantibody against HTTwas produced by immunizing animalswith a synthetic peptide corresponding to residues sur-rounding Pro1220 of human HTTprotein and it is unlikelythat truncated HTT proteins cannot be recognized by thisprimary antibody

Collectively our results suggest that downstream non-canonical ATGs may not be effective in the genomic contextwhich is quite different from their capability in plasmidconstructs Several factors might be accounted for thisdiscrepancy leading to a failed proof of concept to exploitthe possible functions of truncated HTT First transienttransfection of HTT-exons-GFP constructs basically resultsin really high-level expression of proteins although we onlytransfected small amounts of plasmids Also CMV is a verystrong promoter in constructs that may accelerate thismassive expression and lead to the formation of cytoplasmicpolyQ aggregates Notably the translated WT polyQs arewithin truncated peptides but not within the full-lengthHTTprotein In this truncated context the conformation oftranslated peptides harboring WT polyQ might be changedand leads to form aggregates more easily On the contrarytransfection of the E3a∆-GFP construct with this strongpromoter only produced small amount of proteins 2ispartially suggests that E3a-ATG is indeed far less efficientthan the canonical E1-ATG start codon Similarly in thenative genomic context noncanonical E3a-ATG also has farlower efficiency to produce enough truncated proteins to bediscoverable by the western blot assay and in this case the


potency of truncated HTT would also be far below thera-peutic requirements Second the complexity of endogenousregulatory system flanking N-terminal HTT is really dif-ferent from the simple well-known plasmid elements Amore comprehensive analysis of endogenous regulatoryelements and their orchestration need to be considered

By using the knock-in technology with the CRISPRsystem further studies may perform gene engineering tokeep the strong E1-ATG after 5primeUTR that directly ligateswith downstream in-frame HTT sequences excluding CAG

repeats Alternatively it might be possible to drive trans-lation from the E3a-ATG by adding some upstream regu-latory elements And for sure pathogenic N-terminal HTTcan also be replaced by a WT fragment with the similarknock-in method to correct the mutations 2e concern isthat the efficacy of knock-in mediated by homologous re-combination (HR) is much lower than that by non-homologous end joining (NHEJ) For in vivo gene targetingor gene therapy it may not be optimal to deliver a stretch ofDNA donor together with CRISPR vectors In this study we

E6∆GFP 405kDa 1 MELFLLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIKKNGAPRSLRAALW

E4∆GFP 324kDa 1 MELFLLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIK------------------------

E3b∆GFP 300kDa 1 MELFLLCSDDAESDVRMVADECLNKVI----------------------------------------------------------

E3a∆GFP 286kDa 1 MELFLLCSDDAESDVR------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------

E6∆GFP 405kDa RFAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESVQETLAAAVPKIMASFGNFANDNEI

E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------

E6∆GFP 405kDa 121 KVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT


E3b∆GFP 300kDa 28 KVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT

E3a∆GFP 286kDa 17 --VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT

GFP 268kDa 1 --VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT

E6∆GFP 405kDa 181 LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL


E3b∆GFP 300kDa 88 LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

E3a∆GFP 286kDa 76 LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

GFP268kDa 60 LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

E6∆GFP 405kDa 241 VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA


E3b∆GFP 300kDa 148 VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA

E3a∆GFP 286kDa 136 VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA

GFP268kDa 120 VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA

E6∆GFP 405kDa 301 DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKlowast


E3b∆GFP 300kDa 208 DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKlowast

E3a∆GFP 286kDa 196 DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKlowast

GFP 268kDa 180 DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKlowast

61

Figure 2 Translation products of HTT GFP constructs initiated by E3a-ATG Translation ORFs were aligned using EMBL-EBI multiplesequence alignment program (T-Coffee httpwwebiacukToolsmsatcoffee) and viewed by BoxShade Server (httpsembnetvital-itchsoftwareBOX_formhtml)


5rsquoUTR Intron 1PolyQ

sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6

sgHTT-7sgHTT-8 sgHTT-9E1-ATG

PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)

CGTGCCGGGCGGGAGACCGCCATGGCGAC CCGGCTGTGGCTGAGGAGCCGCTGCACCGACCpolyQsgRNA-4 sgRNA-9

5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240

C GGGA GA C C G G TG GC T GA G GA GC C GC TG CAC C GA C C G T GA G T T TG GGC C C G

C GGG A GA C C G G TG GC TG A GG A GC C GC TGC AC C GA C C A AA G AA A GAAC T T T C

(d)

Figure 3 Continued


originally aimed to figure out noncanonical ATGs that mayinitiate truncated HTTtranslation in which case we can turndetrimental HTTdirectly into functional ones only by NHEJmechanisms Since E3a-ATG appears not to be strongenough in the genomic context these knock-in strategiescurrently may not be promising for in vivo gene therapyHowever these generated knock-in cells would be helpfulfor future studies in dissecting truncated HTT functionsOverall our study examined the therapeutic efficacy ofdownstream noncanonical ATGs for HTT translation andalso provided an optimized dual-sgRNA strategy for furthergenome manipulation of the HTT gene

Data Availability

2e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

2e authors declare that there are no conflicts of interest

Authorsrsquo Contributions

YT and CLZ conceived this study JW and YTperformed allthe experiments JW and YT prepared and revised the

manuscript All authors reviewed and approved the finalmanuscript

Acknowledgments

2is study was supported by grants from the NationalNatural Science Foundation of China (No 81801200) andHunan Provincial Natural Science Foundation of China (No2019JJ40476)

References

[1] H M Yang S Yang S S Huang B S Tang and J F GuoldquoMicroglial activation in the pathogenesis of Huntingtonrsquosdiseaserdquo Frontiers in Aging Neuroscience vol 9 p 193 2017

[2] J M Burgunder ldquoTranslational research in Huntingtonrsquosdisease opening up for disease modifying treatmentrdquoTranslational Neurodegeneration vol 2 no 1 p 2 2013

[3] E Cattaneo C Zuccato and M Tartari ldquoNormal huntingtinfunction an alternative approach to Huntingtonrsquos diseaserdquoNature Reviews Neuroscience vol 6 no 12 pp 919ndash930 2005

[4] H Jiang Y M Sun Y Hao et al ldquoHuntingtin gene CAGrepeat numbers in Chinese patients with Huntingtonrsquos diseaseand controlsrdquo European Journal of Neurology vol 21 no 4pp 637ndash642 2014

[5] I Han Y You J H Kordower S T Brady and G A MorfinildquoDifferential vulnerability of neurons in Huntingtonrsquos disease

WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )

Figure 3 Targeting N-terminal HTT with a dual-sgRNA strategy (a) Design of sgRNAs flanking the polyQ region (b) Detection ofcleavage efficiencies with different combinations of sgRNA pairs by the T7E1 assay Mixed indels were calculated based on the fractions ofPCR products ldquoardquo represents the undigested PCR product and ldquobrdquo and ldquocrdquo represent cleavage products (c) Single colonies screening byjunction PCR after sgHTT-49 targeting (d) Sanger sequencing results of gDNA and cDNA junctions in the sgHTT-49-7 cell colony(e) Measurement of HTT levels by quantitative PCR (f ) Western blot detection of full-length HTTand possible truncated HTTproteins


the role of cell type-specific featuresrdquo Journal of Neuro-chemistry vol 113 no 5 pp 1073ndash1091 2010

[6] S E Andrew Y Paul Goldberg B Kremer et al ldquo2e re-lationship between trinucleotide (CAG) repeat length andclinical features of Huntingtonrsquos diseaserdquo Nature Geneticsvol 4 no 4 pp 398ndash403 1993

[7] F Saudou and S Humbert ldquo2e biology of huntingtinrdquoNeuron vol 89 no 5 pp 910ndash926 2016

[8] X Feng S Luo and B Lu ldquoConformation polymorphism ofpolyglutamine proteinsrdquo Trends in Biochemical Sciencesvol 43 no 6 pp 424ndash435 2018

[9] R K Graham Y Deng E J Slow et al ldquoCleavage at thecaspase-6 site is required for neuronal dysfunction and de-generation due to mutant huntingtinrdquo Cell vol 125 no 6pp 1179ndash1191 2006

[10] J Gafni E Hermel J E Young C L WellingtonM R Hayden and L M Ellerby ldquoInhibition of calpaincleavage of huntingtin reduces toxicityrdquo Journal of BiologicalChemistry vol 279 no 19 pp 20211ndash20220 2004

[11] A Lunkes K S Lindenberg L Ben-Haıem et al ldquoProteasesacting on mutant huntingtin generate cleaved products thatdifferentially build up cytoplasmic and nuclear inclusionsrdquoMolecular Cell vol 10 no 2 pp 259ndash269 2002

[12] J P Miller J Holcomb I Al-Ramahi et al ldquoMatrix metal-loproteinases are modifiers of huntingtin proteolysis andtoxicity in Huntingtonrsquos diseaserdquo Neuron vol 67 no 2pp 199ndash212 2010

[13] A L Southwell C W Bugg L S Kaltenbach et al ldquoPer-turbation with intrabodies reveals that calpain cleavage isrequired for degradation of huntingtin exon 1rdquo PLoS Onevol 6 no 1 Article ID e16676 2011

[14] Z Zheng A Li B B Holmes J C Marasa andM I Diamond ldquoAn N-terminal nuclear export signal reg-ulates trafficking and aggregation of Huntingtin (Htt) proteinexon 1rdquo Journal of Biological Chemistry vol 288 no 9pp 6063ndash6071 2013

[15] X Gu J P Cantle E R Greiner et al ldquoN17 Modifies mutanthuntingtin nuclear pathogenesis and severity of disease in HDBAC transgenic micerdquo Neuron vol 85 no 4 pp 726ndash7412015

[16] R S Atwal J Xia D Pinchev J Taylor R M Epand andR Truant ldquoHuntingtin has a membrane association signalthat can modulate huntingtin aggregation nuclear entry andtoxicityrdquo Human Molecular Genetics vol 16 no 21pp 2600ndash2615 2007

[17] L F DiGiovanni A J Mocle J Xia and R Truant ldquoHun-tingtin N17 domain is a reactive oxygen species sensor reg-ulating huntingtin phosphorylation and localizationrdquoHumanMolecular Genetics vol 25 no 18 pp 3937ndash3945 2016

[18] N S Caron C R Desmond J Xia and R Truant ldquoPoly-glutamine domain flexibility mediates the proximity betweenflanking sequences in huntingtinrdquo Proceedings of the NationalAcademy of Sciences vol 110 no 36 pp 14610ndash14615 2013

[19] J S Steffan N Agrawal J Pallos et al ldquoSUMO modificationof Huntingtin and Huntingtonrsquos disease pathologyrdquo Sciencevol 304 no 5667 pp 100ndash104 2004

[20] L M 2ompson C T Aiken L S Kaltenbach et al ldquoIKKphosphorylates huntingtin and targets it for degradation bythe proteasome and lysosomerdquo e Journal of Cell Biologyvol 187 no 7 pp 1083ndash1099 2009

[21] X Cong J M Held F DeGiacomo et al ldquoMass spectrometricidentification of novel lysine acetylation sites in huntingtinrdquoMolecular amp Cellular Proteomics vol 10no 10 Article IDM111009829 2011

[22] M T El-Daher E Hangen J Bruyere et al ldquoHuntingtinproteolysis releases non-polyQ fragments that cause toxicitythrough dynamin 1 dysregulationrdquo e EMBO Journalvol 34 no 17 pp 2255ndash2271 2015

[23] GWang X Liu M A Gaertig S Li and X-J Li ldquoAblation ofhuntingtin in adult neurons is nondeleterious but its depletionin young mice causes acute pancreatitisrdquo Proceedings of theNational Academy of Sciences vol 113 no 12 pp 3359ndash33642016

[24] X Liu C E Wang Y Hong et al ldquoN-terminal huntingtinknock-in mice implications of removing the N-terminalregion of huntingtin for therapyrdquo PLoS Genetics vol 12 no 5Article ID e1006083 2016

[25] E L Pfister L Kennington J Straubhaar et al ldquoFive siRNAstargeting three SNPs may provide therapy for three-quartersof Huntingtonrsquos disease patientsrdquo Current Biology vol 19no 9 pp 774ndash778 2009

[26] M Takahashi S Watanabe M Murata et al ldquoTailor-madeRNAi knockdown against triplet repeat disease-causing al-lelesrdquo Proceedings of the National Academy of Sciencesvol 107 no 50 pp 21731ndash21736 2010

[27] S Aguiar B van der Gaag and F A B Cortese ldquoRNAimechanisms in Huntingtonrsquos disease therapy siRNA versusshRNArdquo Translational Neurodegeneration vol 6 p 30 2017

[28] H B Kordasiewicz L M Stanek E V Wancewicz et alldquoSustained therapeutic reversal of Huntingtonrsquos disease bytransient repression of huntingtin synthesisrdquo Neuron vol 74no 6 pp 1031ndash1044 2012

[29] J B Carroll S C Warby A L Southwell et al ldquoPotent andselective antisense oligonucleotides targeting single-nucleo-tide polymorphisms in the Huntington disease geneallele-specific silencing of mutant huntingtinrdquo Molecular erapyvol 19 no 12 pp 2178ndash2185 2011

[30] M E Oslashstergaard A L Southwell H Kordasiewicz et alldquoRational design of antisense oligonucleotides targeting singlenucleotide polymorphisms for potent and allele selectivesuppression of mutant huntingtin in the CNSrdquo Nucleic AcidsResearch vol 41 no 21 pp 9634ndash9650 2013

[31] A L Southwell H B Kordasiewicz D Langbehn et alldquoHuntingtin suppression restores cognitive function in amouse model of Huntingtonrsquos diseaserdquo Science TranslationalMedicine vol 10no 461 Article ID eaar3959 2018

[32] M Yu Y Fu Y Liang et al ldquoSuppression of MAPK11 orHIPK3 reduces mutant Huntingtin levels in Huntingtonrsquosdisease modelsrdquo Cell Research vol 27 no 12 pp 1441ndash14652017

[33] C Pidgeon and H Rickards ldquo2e pathophysiology andpharmacological treatment of Huntington diseaserdquo Behav-ioural Neurology vol 26 no 4 pp 245ndash253 2013

[34] J W Shin K H Kim M J Chao et al ldquoPermanent in-activation of Huntingtonrsquos disease mutation by personalizedallele-specific CRISPRCas9rdquo Human Molecular Geneticsvol 25 no 20 pp 4566ndash4576 2016

[35] A MMonteys S A Ebanks M S Keiser and B L DavidsonldquoCRISPRCas9 editing of the mutant huntingtin allele in vitroand in vivordquo Molecular erapy vol 25 no 1 pp 12ndash232017

[36] S Yang R Chang H Yang et al ldquoCRISPRCas9-mediatedgene editing ameliorates neurotoxicity in mouse model ofHuntingtonrsquos diseaserdquo Journal of Clinical Investigationvol 127 no 7 pp 2719ndash2724 2017

[37] M Dabrowska W Juzwa W J Krzyzosiak et al ldquoPreciseexcision of the CAG tract from the huntingtin gene by Cas9nickasesrdquo Frontiers in Neuroscience vol 12 p 75 2018


[38] X Xu Y Tay B Sim et al ldquoReversal of phenotypic abnor-malities by CRISPRCas9-mediated gene correction inHuntington disease patient-derived induced pluripotent stemcellsrdquo Stem Cell Reports vol 8 no 3 pp 619ndash633 2017

[39] N Kolli M Lu P Maiti et al ldquoCRISPR-Cas9 mediated gene-silencing of themutant huntingtin gene in an in vitromodel ofHuntingtonrsquos diseaserdquo International Journal of MolecularSciences vol 18 no 4 p 754 2017

[40] Z Hu Z Shi X Guo et al ldquoLigase IV inhibitor SCR7 en-hances gene editing directed by CRISPR-Cas9 and ssODN inhuman cancer cellsrdquo Cell amp Bioscience vol 8 no 1 p 122018

[41] Y Tang M L Liu T Zang et al ldquoDirect reprogrammingrather than iPSC-based reprogramming maintains aginghallmarks in human motor neuronsrdquo Frontiers in MolecularNeuroscience vol 10 p 359 2017

[42] S Brogna and J Wen ldquoNonsense-mediated mRNA decay(NMD) mechanismsrdquo Nature Structural ampMolecular Biologyvol 16 no 2 pp 107ndash113 2009


Hindawiwwwhindawicom Volume 2018

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and TreatmentHindawiwwwhindawicom Volume 2018

Neurology Research International


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Research and TreatmentSchizophrenia

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018Hindawiwwwhindawicom Volume 2018

Neural PlasticityScienticaHindawiwwwhindawicom Volume 2018


Parkinsonrsquos Disease

Sleep DisordersHindawiwwwhindawicom Volume 2018


Neuroscience Journal

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Computational and Mathematical Methods in Medicine

Multiple Sclerosis InternationalHindawiwwwhindawicom Volume 2018

StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018


Behavioural Neurology


Case Reports in Neurological Medicine

Submit your manuscripts atwwwhindawicom

including caspases calpains cathepsins matrix metal-loproteinases and aspartic proteases that cleave HTT havebeen reported [9ndash13]

2e N17 domain as an evolutionarily conserved domainamong vertebrates functions as a nuclear export signal(NES) [14] and is necessary for nuclear exclusion of smallmutant HTT fragments thereby modifying nuclear patho-genesis and disease severity [15] 2e N17 domain forms anamphipathic α-helical structure that signals endoplasmicreticulum (ER) stress [16] and also senses reactive oxygenspecies (ROS) stress specifically through direct oxidation ofa highly conserved methionine at position 8 [17] Moreoverthe polyQ tract can act as a flexible domain allowing theflanking domains to come into close spatial proximity toform proper functional conformations [18] however thisflexibility may be impaired with expanded polyQ tractsImportantly multiple posttranslational modifications suchas phosphorylation ubiquitination acetylation andsumoylation have been identified in the N17 domain thatmay affect the clearance of HTT and its subcellular locali-zation [19ndash21] 2us N-terminal HTT is considered to beimportant in regulating protein aggregation subcellularlocalization and cytotoxicity Despite of this fact othermodification sites in the remaining HTT protein have beendemonstrated to possess pivotal cellular functions as well[7] For instance sequential N-terminal proteolysis of HTTalso generates a C-terminal fragment which induces cyto-toxicity via abnormal vacuolation of ER and increases ERstress [22] Interestingly without a whole stretch ofN-terminus however HTTmay also retain some beneficialfunctions [23 24] For example Liu et al reported that atruncated HTT lacking the first 237 amino acids spanningexon 1sim6 can rescue the HTT deficiency-induced neuriticdegeneration which largely depends on the dynein-bindingregion known to be involved in intracellular trafficking [24]

So far different strategies have been employed to pro-duce therapeutic effects in both cellular and animal modelsof HD including traditional avenues such as RNA in-terference (RNAi) [25ndash27] and antisense oligonucleotides(ASOs) [28ndash31] pharmacological treatments [32 33] andthe emerging genome editing by CRISPRCas9 both in vitroand in vivo [34ndash39] A recent study by Southwell et aldeveloped ASOs targeting HD single-nucleotide poly-morphisms (SNPs) that can selectively suppress mutantHTT expression in the mouse HD model [31] 2ey alsoachieved preclinical efficacy data of those HTT ASOs de-livered to cynomolgus monkeys via lumbar puncture [31]Similarly two other recent studies have used an allele-se-lective CRISPRCas9 strategy based on protospacer adjacentmotif (PAM) sites created by altering SNPs thereby selec-tively inactivating mutant HTT allele for a given diplotype[34 35] As this is a quite novel personalized strategy itrequires a comprehensive analysis of patientsrsquo HTT hap-lotype patterns 2e nonallele-selective inactivation of HTThas also been achieved by using a pair of sgRNAs flankingCAG repeats in a transgenic HD mouse model [36] as wellas in patient-derived fibroblasts with the help of Cas9nickase [37] CRISPRCas9 was also employed to correct theHTT mutation in an HD-induced pluripotent stem cell

(iPSC) line which can rescue the HD phenotypes such asimpaired neural rosette formation increased susceptibilityto growth factor withdrawal and deficits in mitochondrialrespiration [38] Notably CRISPRCas9 can permanentlydisrupt the targeted genes rendering it to be a more efficienttherapeutic approach than those with continuous delivery ofdrugs for treatment However it also brings about the ca-veats that the treatment cannot be reversed in the event ofadverse effects

Taking advantage of the higher efficiency by the CRISPRCas9 system we hypothesize that we may remove a length ofN-terminal HTT to abolish the neurotoxicity stressed bypolyQ aggregations and meanwhile keep the functions ofprobably generated truncated HTT after CRISPR targetingTo successfully obtain this type of endogenous truncatedHTT it is required that an alternative downstream ATGstart codon will be capable of initiating truncated HTTtranslation albeit at lower levels 2erefore in this study wetried to search all possible in-frame ATGs within the first 6exons that coded for 249 amino acids which were thencloned to fuse with GFP in plasmids and we demonstratedthat one of them can indeed start downstream translationHowever after removing endogenous N-terminal HTT byan optimized dual-sgRNA targeting we failed to detectobvious traits of truncated HTT expression Our resultssuggest that noncanonical ATGs of N-terminal HTT maynot be effective in the genomic context as in the constructcontext

2 Materials and Methods

21 Cloning and Sequencing Constructs of E3a- E3b- E4-and E6-GFP were generated based on the pcDNA31(+)vector using Gibson Assembly Master Mix (New EnglandBiolabs MA USA) Specifically different lengths of HTTfragments were amplified from a human cDNA library with21 CAGs located in N-terminus E3a∆- E3b∆- E4∆- andE6∆-GFP plasmids were then respectively constructed byPCR using primers flanking exon 2 and 5primeUTR from theircorresponding plasmids and finally self-ligated into circleplasmids 2e length of 5primeUTR in all constructs is 145 bpwhich is exactly the full-length of 5primeUTR in the NM_002111transcript All pcDNA constructs were finally confirmed bySanger sequencing For genome editing lentiCRISPRv2(Addgene 52961) was used as the backbone and sgHTTsequences were designed with the help of CHOPCHOPwebsite (httpchopchopcbuuibno) In brief sgRNAoligos were annealed and inserted into the BsmBI digestedvector Junctions of exon 12 or targeted genomic regionswere amplified by PCR and ligated into the pMD20T vector(Takara Bio Inc CA USA) for T-A cloning White colonieswere picked out for plasmid extraction and Sanger se-quencing Sequences of sgHTTand PCR primers are listed inTable 1

22 Cell Culture HEK293 cells were maintained in theDMEM medium (2ermo Fisher Scientific MA USA)supplemented with 10 fetal bovine serum (Corning NY




(1 minus fcut)

1113968)










ACCGCCAUGG

AUCGCUAUGG

E1 ATG

E3a ATG

E3b ATG

E4 ATG

E6 ATG

GCUUUGAUGG

GUCAGGAUGG

AAAAUUAUGG

(a)

E6-GFPE4-GFP

E3b-GFPE3a-GFP

E6∆-GFPE4∆-GFP




E2E2E2

E2E2E2E2

E3

E3E3E3E3

E3E3E3

E4E4

E4E4

E5

E5

E6

E6EGFPEGFPEGFP

EGFPEGFPEGFPEGFP


PolyQ

5rsquoUTR EGFP

E2

(b)GFP

E6-G

FPE4

-GFP

E3b-

GFP

E3a-

GFP

610 620 630

530 540

710 720

930 940





(c)

E6∆-

GFP

E4∆-

GFP

E3b∆

-GFP

E3a∆

-GFP

Exon 25rsquoUTR

180 190 200

180 190 200

180 190 200

180 190 200





(d)

GFP

E3a-GFP E3a∆-GFP

E3b-GFP E3b∆-GFP

E4-GFP E4∆-GFP

E6-GFP E6∆-GFP

(e)

E6Δ-GFPE4Δ-GFP

E3bΔ-GFP

E3aΔ-GFP

GFP

α-Tubulin

GFP

43kDa

34kDa

E6Δ-GFP

E4Δ-GFP

E3bΔ

-GFP

E3aΔ

-GFP

26kDa

268kDa286kDa

300kDa

324kDa405kDa

GFP

lowast

lowastlowast

lowast

(f )



















GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------


E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------





















61




sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal












(1 minus fcut)

1113968)










ACCGCCAUGG

AUCGCUAUGG

E1 ATG

E3a ATG

E3b ATG

E4 ATG

E6 ATG

GCUUUGAUGG

GUCAGGAUGG

AAAAUUAUGG

(a)

E6-GFPE4-GFP

E3b-GFPE3a-GFP

E6∆-GFPE4∆-GFP




E2E2E2

E2E2E2E2

E3

E3E3E3E3

E3E3E3

E4E4

E4E4

E5

E5

E6

E6EGFPEGFPEGFP

EGFPEGFPEGFPEGFP


PolyQ

5rsquoUTR EGFP

E2

(b)GFP

E6-G

FPE4

-GFP

E3b-

GFP

E3a-

GFP

610 620 630

530 540

710 720

930 940





(c)

E6∆-

GFP

E4∆-

GFP

E3b∆

-GFP

E3a∆

-GFP

Exon 25rsquoUTR

180 190 200

180 190 200

180 190 200

180 190 200





(d)

GFP

E3a-GFP E3a∆-GFP

E3b-GFP E3b∆-GFP

E4-GFP E4∆-GFP

E6-GFP E6∆-GFP

(e)

E6Δ-GFPE4Δ-GFP

E3bΔ-GFP

E3aΔ-GFP

GFP

α-Tubulin

GFP

43kDa

34kDa

E6Δ-GFP

E4Δ-GFP

E3bΔ

-GFP

E3aΔ

-GFP

26kDa

268kDa286kDa

300kDa

324kDa405kDa

GFP

lowast

lowastlowast

lowast

(f )



















GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------


E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------





















61




sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal










ACCGCCAUGG

AUCGCUAUGG

E1 ATG

E3a ATG

E3b ATG

E4 ATG

E6 ATG

GCUUUGAUGG

GUCAGGAUGG

AAAAUUAUGG

(a)

E6-GFPE4-GFP

E3b-GFPE3a-GFP

E6∆-GFPE4∆-GFP




E2E2E2

E2E2E2E2

E3

E3E3E3E3

E3E3E3

E4E4

E4E4

E5

E5

E6

E6EGFPEGFPEGFP

EGFPEGFPEGFPEGFP


PolyQ

5rsquoUTR EGFP

E2

(b)GFP

E6-G

FPE4

-GFP

E3b-

GFP

E3a-

GFP

610 620 630

530 540

710 720

930 940





(c)

E6∆-

GFP

E4∆-

GFP

E3b∆

-GFP

E3a∆

-GFP

Exon 25rsquoUTR

180 190 200

180 190 200

180 190 200

180 190 200





(d)

GFP

E3a-GFP E3a∆-GFP

E3b-GFP E3b∆-GFP

E4-GFP E4∆-GFP

E6-GFP E6∆-GFP

(e)

E6Δ-GFPE4Δ-GFP

E3bΔ-GFP

E3aΔ-GFP

GFP

α-Tubulin

GFP

43kDa

34kDa

E6Δ-GFP

E4Δ-GFP

E3bΔ

-GFP

E3aΔ

-GFP

26kDa

268kDa286kDa

300kDa

324kDa405kDa

GFP

lowast

lowastlowast

lowast

(f )



















GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------


E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------





















61




sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal


























GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------


E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------





















61




sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal

















GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------


E4∆GFP 324kDa 48 ----------------------------------------------------------------------------------------------------------

E3b∆GFP 300kDa 28 ----------------------------------------------------------------------------------------------------------

E3a∆GFP 286kDa 17 ----------------------------------------------------------------------------------------------------------

GFP 268kDa 1 ----------------------------------------------------------------------------------------------------------





















61




sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal











sgHTT-1sgHTT-2

sgHTT-3sgRNA-4

sgHTT-5sgHTT-6


PolyPN17Exon 1

(a)

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

a ab

ab

abbc

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-1 sgHTT-2

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-3

sgH

TT-5

sgH

TT-6

sgH

TT-7

sgH

TT-8

sgH

TT-9

sgHTT-4

sgEG

FP

Mix

edin

del (

)

279

330

363

357

278 26

34

42

40

76

160

160

202

219

274

abc

abc

(b)

sgHTT-49WT 1 3 4 5 6 7 8 9 10 12

(c)

7 gDNA seq(55)

7 cDNA seq(55)


5rsquoUTR Intron 1

GTGAGT T TGGGCCCG

Intron 1

Exon 2

200210220230240



(d)

Figure 3 Continued



Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal











Data Availability







Acknowledgments


References






WT

sgH

TT-4

9-

6

sgH

TT-4

9-

7

sgH

TT-4

9-

10

Rela

tive m

RNA

expr

essio

n of

HTT

00

02

04

06

08

10

12

(e)

Mice brain lysate

HEKWT

HEKsgHTT-49-7

α-Tubulin

HTT

315k

Da

140k

Da

(f )































































MedicineAdvances in



Psychiatry Journal






































































MedicineAdvances in



Psychiatry Journal




































MedicineAdvances in



Psychiatry Journal






























MedicineAdvances in



Psychiatry Journal










Documents

TargetingN-TerminalHuntingtinwithaDual-sgRNA ...downloads.hindawi.com/journals/bmri/2019/1039623.pdfincluding caspases, calpains, cathepsins, matrix metal-loproteinases,andasparticproteases,thatcleaveHTThave