PiggyBac gRNA Expression Vector

CRISPR/Cas9 vectors are a體姐mong several types of emerging genome 數少editing tools tha術知t can quickly and 鄉玩efficiently create muta作謝tions at target sites of a 離有genome (the other two popular ones bei就雪ng ZFN and TALEN).

Cas9 is a member of 商南a class of RNA-gui她答ded DNA nucleases whic來唱h are part of a natural畫中 prokaryotic immun朋照e system that confers resistance他要 to foreign genetic elements such as pl對男asmids and bacteriopha見件ge. Within the cell, t謝這he Cas9 enzyme fo樂了rms a complex with a guide 術我RNA (gRNA), which provides targ員為eting specificity through 新在direct interaction w知拍ith homologous 18-2的亮2nt target sequences i地和n the genome. Hybridizati到高on of the gRNA to the target site 街水localizes Cas9, which村紅 then cuts the target site in the g懂影enome.

To achieve CRISPR-m作市ediated gene targeting it is essent師歌ial for the target cells t視風o co-express both Ca音站s9 as well as the target site-spec歌制ific gRNA at the 哥有same time. This她說 can be accomplished by either 火歌expressing both Cas9 and the gRN計村A sequence from the same行慢 vector (a.k.a. all-in-one水草 vector) or by using se懂票parate vectors for driving Cas9 and制笑 gRNA expression (Cas9 only區上 and gRNA only vectors respect技間ively). The advantage of using s地嗎eparate vectors over兒船 an all-in-one vector for expres懂飛sing Cas9 and gRNA is that i門司t offers the flexibility of&n銀計bsp;combinatorial usage of different長信 gRNA expression vect理服ors in conjunctio請船n with a variety of Cas9 vari微還ants (wild type nuclease, nickase, nu那的clease-dead) depending upon the user’s紙作 experimental goa子風l. Additionally, using a訊是 separate gRNA only vector all窗能ows cells or organ飛樹isms stably expres為錯sing high levels 舊事of Cas9 to be transf市用ected with different gRNA seque了女nces targeting either the same舊亮 gene or different 分時genes. This provides an opportunity fo來呢r comparing the e拍又fficiencies of different gRNA sequenc那大es in parallel at CRISPR-media匠靜ted gene targeting in c北空ells or organisms with compar內做able and high levels of Cas9 expres如東sion.

The piggyBac gRNA expression vect機刀or is highly effectiv器是e for achieving transfection-media妹個ted permanent introductio銀友n of target sit現東e-specific gRNA sequence into the h分飛ost genome of mammalian cells.業朋 The piggyBac system con會能tains two vectors, both engi可讀neered as E. co訊輛li plasmids. One vector日明, referred to as the help的哥er plasmid, encodes the t鄉見ransposase. The other vector紅少, referred to as物門 the transposon plas關山mid, contains two terminal repeat姐校s (TRs) bracket公花ing the region t書爸o be transposed. The 火放;gRNA expression cassette c還時onsisting of a human員花 U6 promoter driving the targe哥樹t site-spec人來ific gRNA sequence遠去 is cloned into 樹年this region during vector construction水讀. When the helper and tran外明sposon plasmids are co-t算劇ransfected into target cells, the 樹嗎transposase produced from the help報秒er would recognize the two TRs on th中兵e transposon, a放業nd insert the flanked region includ科報ing the two TRs 間我into the host genome. Inserti的計on typically occurs at作坐 host chromosomal sites that呢師 contain the TTAA sequence, which is du著南plicated on the two flanks of the in店工tegrated fragment費女.

Our piggyBac gRNA exp錢月ression vector is avai這拍lable for expres們睡sing either single化電-gRNA or dual-來微gRNAs. While the single-gRNA vecto器車r is widely used for conven睡美tional CRISPR genome 拿要editing applications such as generat報腦ing single gene kno廠市ckouts, dual-gRNA vectors are nec風門essary for applicatio如費ns requiring simultaneous t了劇argeting of a pair of genomic sites.不南 Examples of such ap術姐plications includ件地e: 1) paired Cas9 nickase exp員是eriments where the睡議 “nickase” mutant for做船m (hCas9-D10A) of hCas9 is used in con區雜junction with two gRNAs targeting the t得亮wo opposite strands of a single target 去答site to generate single議小 strand cuts one on each s文都trand, thereby leading近時 to a DSB with i照訊ncreased targeting specific光算ity than a single gRNA; 2)&nb理店sp;generating d冷呢eletion of a fragment be爸如tween two DSBs targeted by a pair of離訊 gRNAs; and 3) targetin的在g two different genes simultaneously. 舞從While the single gRNA vector cons光嗎ists of a single human U6 promot一對er driving the target site-兒家specific gRNA sequence between the習城 two TRs, the dual gRNA vector con器街sists of two consecutive U6 p生司romoters driving the expres廠短sion of gRNA sequences sp廠業ecific to two genomic target sites離技 of interest.

PiggyBac is a class II transposon,光聽 meaning that it m房舞oves in a cut-and-paste manner, h做公opping from place to place without le錢資aving copies behind. (In co海服ntrast, class I transposons mo光年ve in a copy-and-也木paste manner.) Because the helper 明國plasmid is only tran聽雜siently transfected in分山to host cells, it will get lost over 亮煙time. With the loss of the helper p這錯lasmid, the integration of 有用the transposon in the genome家街 of host cells becomes permanent. If th年得ese cells are transfected with 朋月the helper plasmid again, the 數什transposon could get ex能嗎cised from the geno暗為me of some cells, footprint 到嗎free.

For further information ab笑身out this vector弟弟 system, please refer to the pap會得ers below.