Gene Targeting 答了Donor Vector

CRISPR/Cas9 (Clustered Regularly市還 Interspaced Short Palind做店romic Repeats/CRISP會雨R associated protein 9) nuclease ex秒報pression vectors a中湖re among several types of emerging ge理鄉nome editing tool船區s that can quickly and 也算efficiently create mutations at t懂不arget sites of a genome (the othe離農r two popular ones bei哥廠ng ZFN and TALEN).

Cas9 is a member of 煙兵a class of RNA-guided DNA nuc風哥leases which are part of a視司 natural prokaryotic immune道現 system that confers resistance t區讀o foreign genetic elements such as plas年黃mids and bacteriophage. Wit頻路hin the cell, the Cas9 e現朋nzyme forms a complex with a gu也刀ide RNA (gRNA), which pro銀靜vides targeting specificity 紅子through direct interacti服亮on with homologous 18-22nt target sequ愛日ences in the genome. H腦章ybridization of就去 the gRNA to the target site影媽 localizes Cas9, which then cuts the 可暗target site in the genome.

Cas9-mediated cleavage秒習 of the DNA target site ultimately r車門esults in a double-strand break (DSB)街道 which can then西家 be repaired by either of the two follo學校wing repair pathways – t看通he non-homologo你體us end joining (NHE草呢J) pathway or the homol冷資ogy directed repai鐘事r (HDR) pathway. Cellular r明金epair of DSBs by NHEJ is more common a區明nd usually results船看 in small deletions, or m友海ore rarely insertions and女讀 base substitutions. Wh金問en these mutations disr好從upt a protein-coding region (e了美.g. a deletion causing銀輛 a frameshift), the result is a f們市unctional gene knockout. Al河開ternatively, and less effici們湖ently, DSBs can這好 be repaired by homology-directe下機d repair (HDR), using exogenous科事 donor DNA template, which i光明s co-introduced into cells wit資吃h the CRISPR/Cas9 components. This房聽 can result in replacemen得從t of the target ge劇討nomic DNA sequence with sequence from 如熱the donor DNA, genera村生ting either small 的吃targeted base chan她樂ges, such as point mutation靜美s or large sequence 兵筆alterations such as還事 fragment knocki有那n.

Our gene targeting donor vectors錯書 are highly effic就服ient vehicles for delivering exogenou錢朋s donor templates to achieve targ飛土eted insertion of reporte黃白rs, fluorescent tags or o得理ther desired sequences at g湖書enomic sites of inter謝海est. The donor vecto村廠r is designed to contain the desired i低車nsertion sequence flanked by upstream體從 and downstream homology arms (homo校農logous sequences upstream and down工她stream of the target si山煙te of interest).但化 Efficient HDR targeting also requires麗外 the DSB introduced樹能 by Cas9 to be located wit樂購hin a proximity o朋黑f the target site of insertio到化n, ideally within 10-15 bp of the ho草輛mologous arms. Additionally短們, when designing donor 麗木vectors for HDR, it is criti舊有cal to either exclude校睡 or inactivate any 現通PAM sequences in我事 the repair template when presen還嗎t, to prevent Cas9 from di去畫srupting the donor海好 template or the edited genomic l笑得ocus after it has been edited內器 by HDR.

Since undesirable off-target長校 effects are a major drawback愛關 associated with CRISPR genome 黃師targeting, careful d山朋esigning of the target-site specific 西拍gRNA sequences with minimal 近事off-target scores i相子s critical. Add去費itionally, off-targe費海t effects can be further minimized朋下 by using the mutant nickase fo湖道rm (hCas9-D10A) of the standa件線rd humanized hCas9 which generates s姐市ingle-stranded cuts in DNA instead of員短 DSBs. If hCas9-D鄉算10A nickase is used in conjuncti東們on with two gRNAs tar靜舞geting the two oppos算物ite strands of a single targe門金t site, it will generate商謝 single strand 電問cuts on both stran慢家ds, resulting in a DSB at th他亮e target site. This 話暗approach generally reduces off-target e問友ffects of CRISPR/Ca從鄉s9 expression because targe木廠ting by both gRNAs i門風s necessary for DSBs to be generated. N票人icked genomic DNA also frequent坐也ly undergoes HDR, and if exog農為enous template DNA 到西in the form of a donor vector不家 is introduced into the cell a要林long with a targeted hCas9-D10A nickas制城e, then desired seque舞這nce changes in the森藍 genome can be generated u得得sing this approach a個是s well.

For further informa遠玩tion about this vector system, please她去 refer to the papers below.

Our gene targeting donor vect生我ors are designed to a嗎時chieve highly efficient HDR-medi秒去ated insertion of reporters, flu舞風orescent tags or other desired大子 sequence at genomic target 兒明sites of interest. The donor vector 了頻is designed with the desired insertio錯師n sequence flanked by target sit弟路e specific upstream and downstr市低eam homologous sequen做年ces to facilitate e唱那fficient recombin厭河ation following C行現RISPR generated DSBs at the genomi姐跳c target sites. The lengths of the 答現homologous arms are adjusted短影 depending upon the size of th子路e desired edit, with lo話女nger insertions requiring longer arm林見s.

Precise changes: Delivering exogenous repair 女舞templates in the form o你不f gene targeting donor vecto區家rs enables HDR-mediated introducti喝人on of precise sequen雨微ce changes at th到草e genomic target sites 銀門of interest.

Technical simplicity: Our gene targeting donor vectors can be錯飛 delivered to mammali制物an cells by conventional tran章她sfection along wit長我h the target site s知農pecific gRNA sequences and問子 the Cas9 protein for HDR票多-mediated genome edit一一ing. Delivering plasmid vector些服s into cells by conventional tr畫呢ansfection is technically straightfor就個ward, and far easier than virus-bas文錢ed vectors which require the pa湖男ckaging of live vir在黃us.