Regular Plasmid gRNA Expression司飛 Vector

CRISPR/Cas9 vectors are among 服街several types of eme商制rging genome editing to這空ols that can quickly公算 and efficiently cre月是ate mutations at target sites 答劇of a genome (the oth遠靜er two popular 多場ones being ZFN and TALEN).

Cas9 is a member of a class 我員of RNA-guided DNA nucleases which裡村 are part of a natural pro但場karyotic immune system that confe有男rs resistance to foreign gen弟高etic elements such as plasmids an道明d bacteriophage. Within the 愛畫cell, the Cas9 會什enzyme forms a complex with a gui國計de RNA (gRNA), which provides日國 targeting specificity through筆話 direct interaction with懂哥 homologous 18-22nt t他制arget sequences in the討日 genome. Hybridizatio市我n of the gRNA to t事還he target site localizes 視海Cas9, which then cuts the t跳場arget site in the妹林 genome.

To achieve CRIS費物PR-mediated gene targeting it is esse那笑ntial for the tar一個get cells to co-ex子坐press both Cas9 as well as冷你 the target site-spec黃討ific gRNA at the same time. This 樹議can be accomplished by either 如窗expressing both Cas這作9 and the gRNA se家還quence from the same v數舞ector (a.k.a. all-in-one vec討西tor) or by using熱答 separate vector我有s for driving Cas9 and 小和gRNA expression (Cas9 onl弟學y and gRNA only 制問vectors respectively). The advantag公現e of using separate vectors o如人ver an all-in-one vect花頻or for expressing C自年as9 and gRNA is店計 that it offers the flexibility of城聽 combinatorial usage 鄉鐵of different gRNA expres家遠sion vectors in問相 conjunction with a喝謝 variety of Cas9 variants (wild typ照靜e nuclease, nickase, nuclea那林se-dead) depend場刀ing upon the user’s experimental go喝會al. Additionally, using a 道物separate gRNA only vector allow說樹s cells or organ街吃isms stably expressing high levels of服兒 Cas9 to be transfected with different 熱又gRNA sequences tar麗要geting either the sa銀北me gene or different 書場genes. This provides the opportunit美生y for comparing th喝吧e efficiencies of differ區你ent gRNA sequences in paralle錯個l at CRISPR-mediated gene targeting 身吃in cells or organisms with comparabl議月e and high levels of 廠微Cas9 expression.

The regular plasmid gRNA exp在拍ression vector is a highly efficient 上好tool for conventional transfec開湖tion-based delivery 綠聽of target site-specific gRNA sequen哥兵ces into mammalian cells. Delivering p答唱lasmid vectors into mammali什時an cells by conventional拿說 transfection is one 謝現of the most widely used爸道 procedures in 商機biomedical rese爸跳arch. While a number of more sop懂來histicated gene delivery vecto費讀r systems have been developed over t會務he years such as lentiviral v輛業ectors, adenovirus vectors, AAV vecto鄉行rs and piggyBac, conve女得ntional plasmid transfecti雪離on remains the wor科小khorse of gene delivery in m外就any labs. This is largely due to it現煙s technical simplicity as wel費風l as good efficiency in a wide 舞這range of cell types. A 店亮key feature of transfection 明風with regular plas黃劇mid vectors is t票工hat it is transient, with only a黃城 very low fraction of cells stably麗兒 integrating the plasmid in the 路務genome (typically less t又媽han 1%).

Our regular pla呢雜smid gRNA expression vector is availabl喝術e for expressing eit兵用her single-gRNA or dual-gRNA樂睡s. While the single-gRNA vector is wid匠公ely used for con錯裡ventional CRISPR genome ed兒懂iting such as gen們下erating single gene knockout, dual我報-gRNA vectors are necessary for applica機房tions requiring simultaneous targeting國子 of a pair of genomic 你水sites. Examples很地 of such applications include: 1)下飛 paired Cas9 nickase experimen和是ts where the “nickase” mut靜學ant form (hCas9-D10A) of hCas9 is used拿說 in conjunction with two gRNAs targ靜化eting the two opposite 船不strands of a single target site to g信拿enerate single strand cu文通ts one on each strand,作多 thereby leading to a 可笑DSB with increased targeti知些ng specificity than a single gRN很快A; 2) gene明公rating deletion of a fragment betwee睡快n two DSBs targeted by a pair of gR鐘些NAs; and 3) targeting 銀影two different genes si草嗎multaneously. While the 業路single gRNA vector consi生兵sts of a single human U6 pro司黑moter driving the target si行資te-specific gRNA sequence, the dual gRN南朋A vector consists 業錯of two consecutiv司區e U6 promoters吧懂 driving the expression of gRNA 林習sequences specifi歌道c to two genomic target sites of暗姐 interest.

For further infor答舊mation about this vect事靜or system, please refer to 笑紙the papers below.