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)

SygRNA^® - Synthetic crRNA and tracrRNA for Genome Editing

Background

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was discovered in bacteria, where it functions much like an immune system against invading viruses and plasmid DNA. Short DNA sequences (spacers) from invading viruses are incorporated at CRISPR loci within the bacterial genome and serve as “memory” of previous infections. Reinfection triggers complementary mature CRISPR RNA (crRNA) to find a matching viral sequence. Together, the crRNA and  trans-activating crRNA (tracrRNA) guide CRISPR-associated (Cas) nuclease to cleave double-strand breaks in “foreign” DNA sequences.

The type II prokaryotic CRISPR “immune system” has been engineered to function as an RNA-guided, mammalian genome editing tool that is simple, easy and quick to implement. SygRNA^® (synthetic crRNA and tracrRNA) when combined with Cas9 protein, form ribonucleoprotein (RNP) complexes that target a specific genomic locus.

Direct introduction of Cas9 RNP strengthens and expands the applications of CRISPR genome modification technology by eliminating the possibility of plasmid DNA integration into the host genome. In initial reports, Cas9:sgRNA RNP resulted in efficient genome modification with higher specificity when compared to cells transfected with Cas9 plasmid (Juris et al. 2015, Kim et al., 2014, Lin et al. 2015, Liang et al. 2015). Furthermore, Cas9 RNP technology holds great promise for therapeutic applications including the recent successful generation of knock-in primary human T cells (Schumann et al. 2015).

Figure 1. Three-component Type II CRISPR/Cas9:

• Cas9 Protein
• crRNA
• tracrRNA
   

Protocols

General Considerations

We recommend using your preferred method to introduce nucleic acids into your cells of interest. Sigma-Aldrich provides a variety of transfection reagents, cell culture media and plates, and custom DNA primers for detection of CRISPR-mediated genome editing. For your reference, we have suggested protocols below.

Recommendations

• Assemble SygRNA^®:Cas9 Protein complexes (RNP) on ice, immediately before use.
• In all instances, combine equal molar amounts of crRNA:tracrRNA.
• For transfection of RNP using a transfection reagent, prepare RNP using a ratio between 1:1:1 to 5:5:1 crRNA:tracrRNA:Cas9 protein.
• For nucleofection of RNP, prepare RNP using a ratio between 1:1:1 to 5:5:2 crRNA:tracrRNA:Cas9 protein.

In general, the steps required for successful introduction of Cas9 RNP into cultured and primary cells are:

1. Prepare SygRNA^® crRNA and tracrRNA reagents
2. Prepare cells
3. Assemble Cas9 RNP
4. Transfect cells with RNP
5. Harvest and assay cells

Preparation and storage of SygRNA^® and Cas9 Protein

SygRNA^® Resuspension – Resuspend dried SygRNA^® crRNAs and tracrRNAs to a concentration of 20 µM (20 picomoles per µl) each in 10 mM Tris-containing buffer* of pH between 7 and 8.
 

*For example, combine 1 ml of 1 M Trizma Hydrochloride Solution (Product No. T2663) with 99 ml nuclease-free water (Product No. W4502) (1:100 dilution) to make a 10 mM solution.

SygRNA^® Storage – Store SygRNA^® stock solutions at -20 °C in a non frost-free freezer and avoid multiple freeze-thaw cycles. Store at -70 °C long term.

Cas9 Protein Resuspension

1. Resuspend the lyophilized Cas9 protein with the supplied 50% glycerol solution according to the table below to achieve a concentration of 5 mg/ml (30 pmol/µl).
2. If a lower concentration of Cas9 protein is required, dilute the Cas9 protein with supplied Dilution buffer immediately before use. Store diluted protein on ice, up to 6 hours.

Cas9 protein storage – Aliquot Cas9 protein solution and store at -20 °C in a non frost-free freezer for up to one month.  Store at -70 °C long term.

Preparation of Cells

Approximately 18-24 hours before use, plate cells in complete growth medium. For most cell types, cultures should be 50-80% confluent at the time of transfection.

Preparation of SygRNA^® RNP and transfection with TransIT-CRISPR^®

6-well plate protocol:
Prepare TransIT-CRISPR:SygRNA^® RNP immediately before transfection

• Warm TransIT-CRISPR Transfection Reagent to room temperature and vortex gently.
• Pipet 1.5 to 15 µl each of 20 µM SygRNA^® crRNA and tracrRNA stock solutions to sterile tube on ice.
• Add 1 to 2 µl of Cas9 protein (30 to 60 pmol) to tube containing synthetic crRNA and tracrRNA. Gently pipet up-and-down to mix.  Incubate on ice 30 minutes for complex formation.
• Add 250 µl of serum-free or reduced serum medium to Cas9 RNP.
• Add 5 - 6.25 µl of TransIT-CRISPR reagent to Cas9 RNP. (Optimized in U2OS cells) The ratio of Cas9 RNP to TransIT-CRISPR reagent may need to be optimized for each cell type.
• Pipet up-and-down gently to mix.
• Incubate at room temperature for 15 – 30 minutes to allow transfection complex formation.

Distribute transfection complexes to cells in complete growth medium

• For each sample, distribute TransIT-CRISPR^®:SygRNA^® RNP drop-wise throughout well.
• Gently rock culture vessel back-and-forth and from side-to-side to distribute TransIT-CRISPR:SygRNA^® RNP complexes.
  Incubate cells 24-72 hours before harvest for assay. It is not necessary to replace the medium.

Additional information can be found at the TransIT-CRISPR technical information page.
*TransIT is a registered trademark of Mirus Bio LLC.

Preparation and Nucleofection of SygRNA^® RNP

12-well plate protocol:
Prepare Nucleofector^® Solution and Cells

• Prepare Nucleofector Kit reagents according to manufacturer’s instructions.
• Using a 12-well plate as an example: obtain enough cells for approximately 250k cells per well in a 12-well plate (final volume per well will be 1 ml).
• Concentrate the cells by centrifugation and remove the medium by aspiration.
• Resuspend cells in Nucleofector Solution, with supplement added, at a volume that will allow the distribution of 100 µl of solution per well.
• Place 0.5 ml of Complete Medium to each well of a 12-well plate.

Prepare SygRNA^® RNP

• Dilute SygRNA^® crRNA and tracrRNA to a 10 µM working solution using a 10 mM Tris-containing buffer* of pH between 7 and 8.
• Pipet 0.6 to 7.5 µl (6 pmol to 75 pmol) of each RNA to sterile tube on ice.
• Dilute Cas9 protein to 1 mg/ml using the supplied Dilution buffer and store on ice.
• Pipet 1 to 5 µl (6 pmol to 30 pmol) of Cas9 protein to the synthetic crRNA and synthetic tracrRNA, mix gently, and incubate at room temperature for 5 minutes. The final volume of SygRNA^® crRNA and tracrRNAplus Cas9 protein should be less than 20 µl.

Nucleofect SygRNA^® RNP

• Pipet 100 µl of resuspended cells in Nucleofector solution to the tube containing RNA and Cas9 protein and pipet gently to mix completely.
• Transfer cell/RNP complex suspension to a certified cuvette.
• Select the appropriate Nucleofector Program and process cells according to manufacturer’s directions.

Distribute nucleofected cells to each well

• Immediately add 400 µl of complete medium to the cuvette and gently transfer the sample into the appropriate well of the prepared 12-well plate. Use the pipettes supplied with the kit and avoid repeated aspiration of the sample.
• Allow cells to grow for 24-72 hours before harvesting for assay. It is not necessary to replace the medium.

Figure 2. Outline of delivering Cas9 RNPs

Figure 3. Modification of the AAVS1 locus by SygRNA^® crRNA and tracrRNARNPs
K562 cells were nucleofected with RNPs composed of synthetic tracrRNA and AAVS1-targeting synthetic crRNA (combined at 1:1 molar ratio) plus 1 µg or 5 µg of Cas9 protein. Cel-1 mutation detection of indels at the AAVS1 locus was performed 48 hours post-transfection.

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