繼ZFN（Zinc Finger Nucleases）技術(shù)后，Merck在2013年推出新一代基因組編輯工具--CRISPR/Cas9，讓研究人員以更快、更經(jīng)濟的方式實現(xiàn)基因組特定位點的編輯。憑借過去10年在基因組編輯領(lǐng)域的豐富經(jīng)驗積累以及專業(yè)的生物信息學(xué)平臺，Merck已經(jīng)成功設(shè)計出覆蓋人類，小鼠和大鼠三個物種的所有基因的CRISPR/Cas9載體，并可以提供在線定制服務(wù)，以及完整的CRISPR實驗workflow解決方案。此外，默克與Sanger Institute合作開發(fā)了人、小鼠全基因組CRISPR 文庫，以幫助科學(xué)家實現(xiàn)基因功能的快速篩選、規(guī)?；哪Ｐ徒⒁约八幬镒饔煤Y選等。

• 高效：優(yōu)化的載體設(shè)計，大限度提高轉(zhuǎn)染效率，簡化篩選工作
• 特異：特殊的gRNA設(shè)計和雙切口酶系統(tǒng)，大限度提高特異性
• 全面：產(chǎn)品齊全，可提供質(zhì)粒、RNA、慢病毒載體、RNP等形式，涵蓋人、大鼠、小鼠、植物等多個物種，更有Sanger Arrayed和Broad Pools全基因組文庫以及重要通路的亞文庫
• 掌控：強大的慢病毒全基因組文庫可輕松進行高通量篩選，全面掌控人或小鼠的基因組

CRISPR/Cas9 基因編輯工具

• Sanger Arrayed Lentiviral CRISPR   Libraries • Lentiviral CRISPR Pools Libraries • CRISPR/Cas9單載體表達系統(tǒng) • CRISPR Cas9-D10A雙切口酶系統(tǒng) • SygRNA® Cas9 RNP系統(tǒng) • CRISPR/Cas9基因激活表達載體 • CRISPR/Cas9在植物中的應(yīng)用 • Cas9蛋白 • CRISPR對照 (DNA and Virus)

Genome Editing in Plants with CRISPR/Cas9

Successful ZFN-induced gene targeting was published as early as 2003. Since that time targeted genome editing technology has rapidly advanced and been made commercially available. Most recently, the discovery of the CRISPR/Cas9 pathway has accelerated interest in this field, opening up new possibilities for research and development. Although the CRISPR pathway was identified in bacteria as part of a putative adaptive immune system, it was quickly adapted to the purpose of modifying eukaryotic genomes. While tools like ZFNs laid the groundwork for genome editing today, there are limitations to this founding technology and others like it: the protein:DNA interaction of ZFNs makes designing them complex, the assembly of the ZFN expression construct is time-consuming, and the options for ZFN targeting are limited in many A-T rich plant genomes. The CRISPR pathway, as it has been adopted and modified for eukaryotic genome editing, overcomes many of these hurdles: it relies on an RNA:DNA interaction to find its genomic target, the recognition sequence required for this binding event is an easily altered 18-20 base pairs, and the only requirement for the nuclease binding is the presence of an NGG next to the target site. The first reports of the use of CRISPR/Cas9 in plants studying transient expression assays using Agrobacterium came out in 2013. The CRISPR/Cas9 technology has been successfully applied in model plants (Nicotiana benthamiana, Arabidopsis thaliana) and crops (rice, wheat) and the list is growing.

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On this page:

• CRISPR/Cas9: What is it and how does it work?
• Benefits of using CRISPR/Cas9 for genome-editing
• Simplified Workflow: CRISPR/Cas9 in Plants
• Ready-to-Use Cas9 and Guide RNA (gRNA) Expression Plasmids for Monocots and Dicots
• Custom CRISPR/Cas9 Plant Products
• Custom CRISPR/Cas9 Order Form

CRISPR/Cas 9: What is it and how does it work?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The discovery of the type II prokaryotic CRISPR “immune system” has allowed for the development for an RNA-guided genome editing tool that is simple, easy and quick to implement. The CRISPR/Cas9 system consists of a single monomeric protein and a chimeric RNA. A 20-nt sequence in the gRNA confers sequence specificity and cleavage is mediated by the Cas9 protein. Watson–Crick base pairing with the target DNA sequence is the basis for gRNA-based cleavage, making sophisticated protein engineering for each target unnecessary. Only a 20 nt in the gRNA is needs to be modified to facilitate the recognize a different target. 

CRISPR/Cas9 consists of a Cas9 protein, a CRISPR RNA (crRNA), and a trans-activating crRNA ( tracrRNA). In gene editing applications, crRNA and tracrRNA are often fused into a single guide RNA (sgRNA). The ribonucleoprotein invades the target with crRNA guide sequence by forming a 20-bp RNA/DNA hybrid and displacing the opposite DNA strand after it encounters a protospacer adjacent motif (PAM), such as NGG. Cas9 endonuclease subsequently cleaves the complementary DNA strand (target strand) with a HNH nuclease domain and the displaced DNA strand (non-target strand) with a RuvC-like nuclease domain to create a double strand break (DSB). The repair of the DSB by host cell via non-homologous end joining (NHEJ) or homology directed repair (HDR) pathways can be utilized to create gene knockout or introduce a specific genetic modification through homologous recombination with a DNA donor.

RNA-guided endonucleases (RGENs) consisting of the Cas9 protein derived from Streptococcus pyogenes and guide RNAs (gRNAs) can be customized by replacing only the RNA component leading to decreases in labor and time compared to other gene editing methods. Using either Agrobacterium tumefaciens or by trans­fecting plasmids that encode them, programmable nucleases can be delivered into plant cells, where these nucleases cleave chromosomal target sites in a sequence-dependent manner.  The result is site-specific DNA double-strand breaks (DSBs) whose repair by endogenous systems results in targeted genome modifications.

Benefits of using CRISPR/Cas9 for genome-editing

Main advantages of CRISPR/Cas9 are in terms of simplicity, accessibility, cost and versatility.

CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene.

Only 20 nt in the gRNA sequence need to be changed to confer a different target specificity, which means that cloning is also unnecessary.

Any number of gRNAs can be produced by in vitro transcription using two complementary annealed oligonucleotide. This allows the inexpensive assembly of large gRNA libraries so that the CRISPR/Cas9 system can be used for high-throughput functional genomics applications.

Another advantage of CRISPR/Cas9 compared to ZFNs and TALENs is the ease of multiplexing. The simultaneous introduction of DSBs at multiple sites can be used to edit several genes at the same time.  It can be particularly useful to knock out redundant genes or parallel pathways. Researchers can engineer large genomic deletions or inversions by targeting two widely spaced cleavage sites on the same chromosome. Multiplex editing with the CRISPR/Cas9 system simply requires the monomeric Cas9 protein and any number of different sequence-specific gRNAs. In contrast, multiplex editing with ZFNs or TALENs requires separate dimeric proteins specific for each target site.

CRISPR/Cas9 system can cleave methylated DNA in human cells allowing genomic modifications that are beyond the reach of the other nucleases. While this has not been specifically explored in plants, it is reasonable to believe that the ability to cleave methylated DNA is intrinsic to the CRISPR/Cas9 system and not dependent on the target genome.
 

Simplified Workflow: CRISPR/Cas9 in Plants

Plant Biotechnology is entering a new era with the introduction of genome editing technologies that enables precise manipulation of specific genomic thereby superseding older methods of random mutagenesis as EMS mutagenesis and g-radiation sequences. Plant CRISPR/Cas9 products are intended for Agrobacterium-mediated plant transformation or biolistic microparticle bombardment or protoplast transformation. The products are based on the type IIA CRISPR/Cas9 derived from Streptococcus pyogenes. The native Cas9 coding sequence was codon optimized for expression in monocots and dicots, respectively. The monocot Cas9 constructs contain a monocot U6 promoter for sgRNA expression, and the dicot Cas9 constructs contain a dicot U6 promoter. The plant selection markers include hygromycin B resistance gene, neomycin phosphotransferase gene, and the bar gene (phosphinothricin acetyl transferase).

The pipeline of generating a CRISPR/Cas9-mutagenised plant line. c, control; m, mutagenized; RE, restriction enzyme. CELI and T7 are DNA endonucleases used in the surveyor assay.
 

Ready-to-Use Cas9 and Guide RNA (gRNA) Expression Plasmids for Monocots and Dicots

• Sigma plant CRISPR/Cas9 products are intended for Agrobacterium-mediated plant transformation or biolistic microparticle bombardment or protoplast transformation
• A codon optimized Cas9 protein and a gRNA are expressed from a single vector and provided as ready-to-use, transfection-grade DNA.

Basic structure of CRISPR/Cas9 constructs for Agrobacterium-mediated Transformation.
 

Basic structure of CRISPR/Cas9 constructs for biolistics or protoplast Transformation.

Ordering CRISPR/Cas9 Vector

To customize and purchase CRISPR click ORDER CRISPR below.

Plant CRISPR/Cas9 Product List

Custom CRISPR GUS vectors

 References

• Li JF, Zhang D, Sheen J. Targeted Plant Genome Editing via the CRISPR/Cas9 Technology. Methods Mol Biol. 2015;1284:239-55.
• Ali Z, Abul-Faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes NJ, Voytas DF, Dinesh-Kumar S, Mahfouz MM: Efficient Virus-Mediated Genome Editing in Plants using the CRISPR/Cas9 System.Mol Plant. 2015 Mar 5. pii: S1674-2052(15)00162-8.
• Luisa Bortesi and Rainer Fischer: The CRISPR/Cas9 system for plant genome editing and beyond Biotechnology Advances Volume 33, Issue 1, January–February 2015, Pages 41–52.
• Li JF, Zhang D, Sheen J. Targeted Plant Genome Editing via the CRISPR/Cas9 Technology. Methods Mol Biol. 2015;1284:239-55.
• Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14:327.
• Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S: Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 2013, 31:691–693.
• Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C: Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 2013, 31:686–688.
• Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J: Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 2013, 31:688–691.
• Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu JK: Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 2013, 23:1229–1232.
• Xie K, Yang Y: RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant. Volume 6, Issue 6, November 2013, Pages 1975–1983.
• Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ: Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 2013, 23:1233–1236.-