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 FAQ、相关产品、参考文献

FAQ

Q: How many sgRNA should I construct for a genome editing project?
A: Due to the unpredictable efficacy of a particular guide RNA construct, for optimal results, We recommend 3 and more sgRNAs to be designed for a specific genome sequence. By designing several constructs, one has increased chances of finding a construct(s) to cleave target DNA with the highest efficiency.

Q: How to design the 20 bp target-specific sequence?

A: We recommend using online tools such as http://crispr.mit.edu/ to design sgRNA candidates. To avoid off-target effects, we recommend searching the genome for the 14-nt specificity region consisting of the 12-nt seed region of the sgRNA and 2 of the 3-nt (NGG) PAM of the genome. Any sgRNA designed with more than one binding site should be discarded.

Q: What is the advantage of SSA assay in screening sgRNA in vitro?

A:Theassay has several features that make it well suited as an initial testofsgRNA activity. First, the assay is performed in the biologicallyrelevant environment of a human cell (or could be any easily transfectedcell type). Adverse events, such as cytotoxicity due to off-targetcleavages, will result in the death of transfected cells. By SSA assay, lossof firefly luciferase signal due to toxicity-dependant cell death canbe distinguished from poor sgRNA activity by the inclusion of aRenilla luciferase control plasmid. Second, there are many specialchallenges to the success of a sgRNA at an endogenous target site,including limited chromatin accessibility, competing endogenousbinding factors, and unexpected local DNA structures. If sgRNA activity is not observed at an endogenous site, knowing that the sgRNA was functional in an SSA assay will help to differentiatecomplications at the endogenous site from problems with the sgRNA. Third, because it is difficult to anticipate the challenges atthe endogenous site, a pragmatic approach would be to create 3–10 sgRNA that target different sites in the region of interest.The SSA assay can be easily reconfigured with a new sgRNAtargetsite with a single oligonucleotide.

Related CRISPR-CAS9 System Products


1) DNA/RNA oligo synthesis
2) sgRNA design and expressing plasmid construct
3) pSD-n- gRNA (negative control)
4) pSD-p-gRNA (positive control)
5) TALEN positive control and SSA Target report plasmid
6) sgRNA design and synthesis
7) luciferase report plasmid pSG-target for validation of crispr-cas9 system
8) sgRNA library and screening


参考文献:


1. Sampson TR, Saroj SD, Llewellyn AC et al: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 2013, 497(7448):254-257.

2. Qi LS, Larson MH, Gilbert LA et al: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152(5):1173-1183.

3. Wang H, Yang H, Shivalila CS et al: One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 2013.

4. Shen B, Zhang J, Wu H et al: Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell research 2013, 23(5):720-723.

5. Chang N, Sun C, Gao L et al: Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell research 2013, 23(4):465-472.

6. Hwang WY, Fu Y, Reyon D et al: Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 2013, 31(3):227-229.

7. Mali P, Yang L, Esvelt KM et al: RNA-guided human genome engineering via Cas9. Science 2013, 339(6121):823-826.

8. Cong L, Ran FA, Cox D et al: Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339(6121):819-823.

9. F. Ann Ran, Patrick D. Hsu, Chie-Yu Lin et al: Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.cell, 2013,154(6):1380-9.

10. Tim Wang,Jenny J. Wei,David M. Sabatini,Eric S. Lander. Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science Vol. 343 . 80-84

11. Ophir Shalem, Neville E. Sanjana, Ella Hartenian, Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells, Science Vol. 343 . 84-87

 

 

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