CRISPR產品和服務

CRISPR/Cas9技術已發展成為基因組編輯的熱門工具，它能夠完成RNA導向的DNA識別及編輯，為更高效的基因編輯技術提供了全新的平臺。除用于基因組編輯，CRISPR技術也應用于眾多其它研究中。作為合成生物學及基因組編輯的行業領先者，金斯瑞可為客戶提供GenCRISPR™產品和服務，此類產品及服務得到美國麻省理工學院Broad研究所張鋒實驗室及哈佛大學授權許可。

CRISPR/Cas9服務

CRISPR/Cas9技術應用

• 基因編輯細胞系構建 – 構建knock-out或knock-in細胞系，用于表型分析或藥物篩選等
• 動物模型 – 利用CRISPR/Cas9已建立轉基因斑馬魚、小鼠和猴子等基因改造的動物模型
• 農業研究 –用于農作物基因功能研究，改良作物的抗旱及抗病原性，可避免引入外源DNA
• 個性化治療 – 從病毒感染者的細胞中去除外源病毒的DNA，治療HIV及其它傳染性疾病
• 基因組調節 – dead Cas9誘導調控靶基因的轉錄或重新組裝DNA

CRISPR-Cas9應用于基因編輯：

• 基因修復--很可能治愈遺傳性疾病和危險因素
• 基因中斷--修正顯性失活突變和入侵的病原體基因組
• 基因抑制--抑制癌基因或宿主病毒受體，防止病毒感染
• 基因激活--抑制腫瘤生長

CRISPR FAQs

Have questions about CRISPR/Cas9? Learn more about the advantages of CRISPR and how to integrate it into your research.

1. What are the advantages of CRISPR gene editing? Read More ?


Of the other gene editing technologies available, CRISPR/Cas9 has stood out for its simplicity and efficacy. The CRISPR system requires only a few simple DNA constructs to encode the gRNA and Cas9, and if knock-in is being performed, the donor template for HR. As a result, CRISPR gene editing is an approachable technique for use in any lab regardless of molecular biology expertise. The table below outlines a few of the key differences between CRISPR gene editing and other popular techniques.



2. How does CRISPR/Cas9 modify eukaryotic genomes? Read More ?








Once Cas9 nucleases are guided to the target DNA and create a double strand break 3-4 bases upstream from the PAM sequences, there are two ways the double strand break (DSB) can be repaired. If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel that effectively knocks out protein function. Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.

3. How are CRISPR reagents delivered to cells? Read More ?


The most efficient method to deliver Cas9 and gRNA plasmids depends largely on the cell type. For easy-to-transfect cell lines, non-viral constructs are often suitable and can be delivered with high efficiency by lipofection. The plasmids usually contain selection markers to confirm effective delivery, such as antibiotic resistance gene s or fluorescent proteins. For hard-to-transfect cell lines, such as stem cells, viral-based transfection methods may be more effective. Lentiviral vectors may be more suitable for these cell types. All of GenScript's lentiviral vectors are compatible with 3rd and 4th generation lenti-packaging systems.
For detailed information on experimental design, we recommend consulting Ran et al's publication:
Ran et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 2013; 8:2281.


4. How efficient and specific is CRISPR-mediated genome editing with gRNA constructs designed by GenScript? Read More ?



CRISPR/Cas9 mediated genome editing is the most efficient and specific form of genome editing used to date. GenScript's scientists have extensive experience designing gRNA sequences for highly efficient KO in numerous types of cell lines. Factors that can affect CRISPR targeting efficiency and specificity are:

• gRNA design: GenScript's proprietary gRNA design algorithm uses the most current genome assembly data available from NCBI and other publicly available sources, and selects the best target sequences to avoid off-target effects. We search for an ~20 bp locus in the endogenous genome of interest for which a highly-similar match does not appear elsewhere in the genome. Off-target Cas9-mediated cleavage can occur even with up to 3 mis-matches between the gRNA and the endogenous genome, though most papers have reported little to no off-target effects.
• nuclease / targeting strategy: Most researches use the Cas9 nuclease isolated from Streptococcus pyogenes. Cas9 WT induces double-strand breaks that are typically repaired through non-homologous end joining (NHEJ), which introduces small insertions or deletions that lead to frame-shifts and total loss of protein expression. This has proven to be an easy and effective way to introduce phenotypic KO in every cell line and organism attempted to date. Another strategy employs a mutant version of this enzyme, Cas9-D10A (Nickase), which can be used to induce two single-strand breaks flanking a region you want to delete for a more specific or comprehensive knock-out. If you require gRNA sequences for use with a different enzyme, or have other special requests for your CRISPR targeting strategy, please email your request to us.
• number of unique gRNA sequences used: Based on our in-house experience using our design tool to create knock-out cell lines, a single gRNA construct is typically sufficient to knock-out your gene of interest; however, we recommend ordering at least two gRNA constructs per gene that you want to target in order to increase your chance of successful genome editing without off-target effects.

When you order gRNA clones from GenScript, we deliver a sequence-verified plasmid containing all elements required for gRNA expression and genome binding: the U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator. We guarantee sequence accuracy for gRNA clones we deliver; however, given the complexity of creating genomically edited cell lines including transfection and selection, we cannot guarantee the outcome of experiments performed using our gRNA constructs. If you prefer to receive sequence-validated KO or KI cell lines created using CRISPR technology, please refer to our GenCRISPR™ mammalian cell line service.

To learn more, please check out our archived webinar:  Can CRISPR/Cas9 off-target genomic editing be avoided? Ways to improve target specificity.

5. What expression vector should I use? Read More ?



Choosing the vector(s) you'll use to express the two critical components needed for CRISPR/Cas9 genome editing, the guide RNA and the Cas9 nuclease, is an important step in your experimental design. Many researchers prefer to use an all-in-one vector that will drive expression of gRNA and Cas9 in a 1:1 ratio. All-in-one vectors may also contain selection markers, such as fluorescent proteins or genes conferring antibiotic resistance, which can make it easier to isolate desired genome-edited clones. You may prefer to express the gRNA and Cas9 from separate vectors, for example if you want to vary the gRNA:Cas9 ratio, or if you want to screen a pool of gRNAs or use a larger gRNA library.

Want some more information? Check out our CRISPR gRNA construct service FAQ.

6. How can I use CRISPR reagents for genome editing in mammalian cell lines? Read More ?



The following procedure is based on HEK293 cells. To use host cell lines other than HEK293, please follow the instruction from original supplier for cell culture, passing the cells, transfection and subcloning. If you have specific questions about how to adapt this protocol for your needs, we recommend consulting the public online forum for Genome Engineering using CRISPR/Cas Systems.



Experimental outline for knocking out a coding sequence in a mammalian cell line:

• I. Host cell preparation
  

  Culture the host cells (HEK293) in Eagle's Minimum Essential Medium supplied with fetal bovine serum to a final concentration of 10%. Incubate cultures at 37°C.
  Subculture when cell concentration is between 6 and 7 x 104 cells/cm2.
  Seed 4-6x104 cells/cm2 in cell culture plate one day before transfection.
  

• II. Transfection
  

  Transfect gRNA and cas9 into HEK293 cells using standard methods for HEK293 transfection. (Multiple transfection reagents give high transfection efficiency for HEK293 cells, following instructions from suppliers.)

  DNA ratio for the two elements for CRISPR-cas9 system

  	Two vector system	All-in-one vector
  gRNA	+	+
  Cas9	+	+
  Starting Ratio of plasmids	1:1	NA



• III. Analysis of transfected cell pool
  

  2-3 days after transfection, the cell pool can be analyzed directly by Sanger sequencing, NGS (Next Generation Sequencing) and/or Surveyor assay. Here the commonly used methods of Sanger sequencing and Surveyor assay (or T7E1 assay) are briefly introduced.

  1. Sanger sequencing of the target region can detect overlapping peaks at close region of double strand break (DSB), if small insertion or deletion (indel) mutations are introduced.
  2. Surveyor assay (or T7E1 assay) uses enzymes of mismatch-specific DNA endonucleases to detect indel mutations at the targeted loci. By targeting and digesting mismatched heteroduplex double-strand DNA, this assay produces two or more smaller fragments, depending on number of mismatched sites on the region analyzed. These assays can be conducted following instructions from supplier.

• IV. Cloning to obtain isogenic cell lines
  

  Transfected cells can be selected using antibiotic resistance or a GFP reporter if they are present on the Cas9 expression plasmid.*
  Transfected cells (with or without selection) can be plated into 96 well plate at 1 cell/well density for cloning. This procedure can be also conducted using diluted host cell line on 10 cm plate to form colonies, which can be picked up and transferred to 24 well plate for future usage.

  *Note, selection using antibiotic containing medium can induce random integration of the cas9 expression plasmid onto host genome.

• 

  V. Screening for cell lines with desired mutation
  

  After expansion of the clones, the cells in each clone can be analyzed by Sanger sequencing to identify the clones harboring a mutation at the target region. Sequencing trace files will show overlapping peaks at regions where double strand breaks have been repaired by introducing small indels.

• VI. Knock out cell line confirmation
  

  Knock-out cell lines can be confirmed by Western Blot if a specific antibody is available, or through functional assays specific to the gene that was targeted.

CRISPR產品和服務執照許可

• 金斯瑞為客戶提供的GenCRISPR™產品和服務得到美國 Broad 研究所，哈佛大學和麻省理工學院授權許可。GenCRISPR™產品和服務受到US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616及多國同等專利保護。
• GenCRISPR™產品和該服務中生成的試劑只能作為工具用于科學研發目的，不得用于以下用途：
  （1）任何人體或臨床的使用；（2）修改任何人類生殖體系，包括改變人類胚胎或人的生殖細胞的DNA；（3）任何作為獸藥或在牲畜體內的使用；（4）制造、批發、進口、出口、運輸、銷售、許諾銷售、市場營銷及推廣，或其他開發，對人類或動物的測試服務，治療或診斷。
• GenCRISPR™服務使用者和產品的購買者：
  不可轉讓GenCRISPR™產品和該服務中生成的試劑于第三者。GenCRISPR™產品和該服務中生成的試劑只能作為工具用于科學研發目的，不得用于任何商業目的。

客戶應明確知曉上述對試劑用途的限制，并自行承擔因違反該限制而產生的法律責任。