PiggyBac CRISPR Vector

CRISPR/Cas9 vectors are among s為來everal types of emerg行分ing genome editing t兒老ools that can quickly路很 and efficiently create muta嗎服tions at target了學 sites of a genome (the other two popul用吃ar ones being ZFN and TALEN).

Cas9 is a member of a class of RNA音錯-guided DNA nucleases which are pa喝技rt of a natural 上微prokaryotic immune system tha術聽t confers resistance to fore相些ign genetic elements such a說說s plasmids and b從草acteriophage. Within the cell, t北人he Cas9 enzyme forms a complex with a 是短guide RNA (gRNA雜家), which provides t國他argeting specificity through di能呢rect interaction with h山照omologous 18-22nt target seq海鄉uences in the genome. Hybridization of 場商the gRNA to the targe自老t site localizes Cas9, which then c長都uts the target site in the geno章作me.

To achieve CRISPR要明-mediated gene targeting it is esse山短ntial for the ta他樂rget cells to co-express 離報Cas9 and the target site-specific gR河畫NA at the same time從文. This can be accomplished 學好by either expressing both Cas9 a子子nd the gRNA sequence from the s樹我ame vector (a.k.a.&n自兒bsp;all-in-one vector) or b機體y using separate vectors for dr生水iving Cas9 and gRNA expression (Cas麗件9 only and gRNA only vectors, re會但spectively). The advanta嗎費ge of using an all-in-學東one vector for ex得從pressing Cas9 and gRNA is了短 that it provides the山議 opportunity to deliver all要照 the required components for 動跳CRISPR-mediated gene editing to th大得e cell using a single vector wh笑時ich is technically strai書市ght forward. Using separate v關能ectors for expressing Cas9 and gRNA 他腦requires co-transfec少雪tion of the target cells員議 with two separate vectors which can 員有be technically challenging什北 since not all cells視熱 will be transfected with both音都 gRNA and Cas9 vectors simulta舊就neously. An alternative approach for u科北sing separate vectors is to很媽 transfect cells or organisms房飛 stably expressing high-錢遠level of Cas9 with the desired 通關gRNA sequences. However, this method 相國can be considerably懂費 time-consuming and labor intensiv習飛e. Our all-in-one p新爸iggyBac CRISPR vector helps to circumv爸靜ent the mentioned challenges b務間y expressing Cas9 and the desired gR通光NA sequence from a 到師single piggyBac vector.

Our piggyBac 外鐵CRISPR vector is 唱得highly effective for achievi站金ng transfection-mediated permane從遠nt introduction of both Ca工房s9 and the target 商煙site-specific gRN綠路A sequence into the host genom對信e of mammalian cells. The piggyBa笑熱c system contains two vectors, bot愛做h engineered as E. coli plasmids他商. One vector, referred to as 們嗎the helper plasmid, encodes一電 the transposase. The other開村 vector, referred to a通湖s the transposon plasmid, co哥道ntains two term業什inal repeats (T哥放Rs) bracketing 化頻the region to be 書還transposed. The gRNA 光在and Cas9 expression cassette is坐科 cloned into this region during 弟為vector construction. A近文 human U6 promoter dri快村ves the expression of the user數林-selected gRNA sequence, w相如hich directs Cas9 to the DNA光懂 target site of interest. 件秒;When the helper and transpos媽風on plasmids are c少哥o-transfected int鐘東o target cells, the transposase prod金外uced from the hel短村per would recognize the two TR好那s on the transpos好謝on, and insert the flanked region inc如少luding the two TRs into the host geno街業me. Insertion typically唱做 occurs at host chro自術mosomal sites tha音水t contain the TTAA sequenc新微e, which is duplicated on 信裡the two flanks o哥技f the integrated fragm事師ent.

Our piggyBac CRISPR vector is availab子時le for expressing either si村水ngle-gRNA or dual-gRN業區As. While the single紅姐-gRNA vector is widely used for c月東onventional CRISPR genome edit舊船ing applications such as generating sin花老gle gene knockouts,麗物 dual-gRNA vectors are necessar理可y for applications requiring simult空話aneous targeting of a pair of 她公genomic sites. Examples of such appl著吧ications include: 1) paired Cas9 nic木區kase experiments where the “nickas女些e” mutant form 兵嗎(hCas9-D10A) of hCas9 is used in co去男njunction with two g行紙RNAs targeting the two 事金opposite strands of a single target s化拍ite to generate si店是ngle strand cuts one on each str多美and, thereby leading to a DSB wi科區th increased targeting specificity t地鐘han a single gRNA; 2) generatin光醫g deletion of a fragment be西微tween two DSBs targeted b也藍y a pair of gRNAs; and 3) t對要argeting two diffe舊頻rent genes simultaneously. Whil我唱e the single 吧書gRNA vector consists of a si紅靜ngle human U6 pr這看omoter driving the target site-算是specific gRNA seq城嗎uence in between the two T水日Rs, the dual gRNA vector consists of 讀議two consecutive U6 南理promoters driving the expressio頻資n of gRNA sequences老笑 specific to two 照師genomic target sites of in海到terest.

Two variants of Cas9 en制那zyme are available i學靜n our all-in-one piggyBac CR多問ISPR vectors. The standard hu刀可manized Cas9 (hCas9) variant ef他中ficiently generates double-strand日放 breaks (DSBs) at target sites, w木購hile the “nickase” mutant form (鐵可hCas9-D10A) generates only single-st你請randed cuts in DNA. If hCas9-問白D10A nickase is used in conjun謝靜ction with two gRNAs targeting the爸生 two opposite strands of 日草a single target site, then the ni和河ckase enzyme will generate sin東音gle strand cuts on both stran船得ds, resulting in司不 DSBs at the target site (as de自山scribed above). This approach general街好ly reduces off-target effects of CRIS亮話PR/Cas9 expression b行冷ecause targeting by b唱花oth gRNAs is necessary for DSBs to be g道說enerated.

PiggyBac is a class II 愛店transposon, meaning that it moves in a 國制cut-and-paste ma錢南nner, hopping from pla慢媽ce to place without leaving城子 copies behind. (In contrast, class I t學我ransposons move in a copy-and-paste 樹看manner.) Because the helper 讀問plasmid is only transiently 理煙transfected into host c答人ells, it will get lost over ti志麗me. With the loss of the白不 helper plasmid, the integration o子就f the transposon in the genome of ho離說st cells becomes permane外學nt. If these cells are transfec姐靜ted with the helper p筆綠lasmid again, the transposon coul鐘白d get excised f服煙rom the genome of some cells, 年笑footprint free.

For further information about thi們在s vector system, please refer t那北o the papers below廠著.

Our piggyBac CRISPR哥森 vector along wit紙西h the helper plasmid are optimize件了d for high copy number repli快土cation in E. coli, effic見裡ient transfection in歌南to a wide range of targe光公t cells, and high-lev爸樂el expression of th爸窗e transgene carried on the 飛北vector. The pig訊少gyBac CRISPR vector system is desig地厭ned to deliver Cas9 and a 購兵target site-specif看們ic gRNA sequence usi匠場ng a single vector.劇雪 This vector is avai友分lable for expressin她關g either single-gRNA or dua唱服l-gRNAs enabling users to target ei車這ther one or two genomi舞都c target sites of interest dep銀票ending upon their experimental goal.

Simplicity: The simple homology relat喝子ionship between the gRNA an中也d the target makes th資數e CRISPR/Cas9 system conceptua做市lly simple and easy to design.&nbs玩畫p;Our piggyBac CRIS現懂PR vector system is designed f錯吃or delivering both Cas9匠兒 as well as the target site-specific 著少gRNA sequence to mammalian cell可嗎s. This provides the opport外員unity to deliver all the費時 required components for CRISPR-me間外diated gene editing to the tar街從get cells using a single p學車iggyBac vector which is tec媽筆hnically straight 票大forward and less time-consumi聽看ng than using two separate vectors f長月or Cas9 and gRNA de快白livery.

Permanent integration of vect熱哥or DNA: Conventional transfection results微少 in almost entirely transi書身ent delivery of DNA into hos了吧t cells due to the lo自得ss of DNA over time. This p得線roblem is especially promin人慢ent in rapidly divi就村ding cells. In contrast, transfection o能美f mammalian cells with the piggyB通快ac transposon pl鐵店asmid along with明家 the helper pla線錢smid can deliver g秒高enes carried on 時做the transposon permane時對ntly into host cell老討s due to the integration你視 of the transpos我煙on into the host gen和高ome.

Technical simplicity: Delivering plasm購呢id vectors into cells 音謝by conventional transfectio鄉女n is technically straightforwar工事d, and far easier than virus-based ve和有ctors which require t村會he packaging of l會亮ive virus.

Single-gRNA piggyBac CRISPR vector

5' ITR: 5' inverted terminal repeat.事音 When a DNA sequence is flanked by two筆門 ITRs, the piggyBac transpose can reco視視gnize them, and i看可nsert the flanked regi木風on including the two ITRs into th年船e host genome.

U6 Promoter: This drives high level e電短xpression of the downstream gRNA.民信 This is the promo行妹ter of the human U6 snRNA gene跳個, an RNA polymerase III promoter w體可hich efficiently expresses s妹什hort RNAs.

gRNA: Guide RNA compatible with Cas腦業9 derived from 學錯;Streptococcus pyogenes.

Terminator: Terminates tran聽的scription of th微男e gRNA.

CBh promoter: Chicken beta-actin promoter商拍. Drives expression of the downstr訊歌eam Cas9 nuclease.

Cas9 protein: Cas9 variant chosen by user. 短放

SV40 late pA: Simian virus 40 late polyadenyl問西ation signal.&nbs話服p;It facilitates transcriptional t得很ermination of the upstream ORF.理老

CMV promoter: Human cytomegalovirus immed一我iate early promoter. It drives th跳們e ubiquitous expressio家城n of the downst一從ream marker gene.

Marker: A drug selection gene (such as neom街上ycin resistance), a visu水員ally detectable gene (such as EGFP)湖個, or a dual-repo哥體rter gene (such as EGFP/Ne花很o). This allows cells transduced with 短放the vector to be selected and/or vis年人ualized.

BGH pA: Bovine gr明睡owth hormone polyadenylation術場 signal. It facilita遠雪tes transcriptional t商筆ermination of the up術她stream ORF.

3' ITR: 3' inverted terminal repeat中視.

Ampicillin: Ampicillin resistance gene. It allows t著冷he plasmid to be maintained by ampicil暗校lin selection in E. co長筆li.

pUC ori: pUC origin of re時電plication. Plas理那mids carrying this origin exist in麗不 high copy numbers in E. coli.資站