CRISPR is C lustered R & lt egularly the I nterspaced S Hort P alindromic R & lt abbreviation epeats Chinese meaning of regularly spaced clusters of short palindromic repeats.
In nature, CRISPR plays a very important role in the immune system of bacteria. When a virus invades bacteria, the molecular mechanism in the bacteria inserts part of the virus's sequence into the CRISPR site. When the virus invades again, the CRISPR site will be transcribed and processed. The processed CRISPR RNA will form a ribonucleoprotein complex with the Cas protein to "scan" the virus genome. When a fragment that is complementary to CRISPR RNA is found, it will bind, and the Cas protein will be cleaved to achieve the purpose of immunity.
In many genome engineering applications, we use Cas9 protein derived from Streptococcus pyogenes , and CRISPR RNA is replaced by artificially synthesized gRNA. The binding of gRNA to the target site of the genome also depends on the presence of a pre-spacer adjacent motif ( PAM ) located downstream of the target site . Among them, the single strand of DNA where the PAM sequence is located and the single strand of DNA bound by gRNA are two different strands. Cas proteins from different prokaryotes recognize different PAM sequences. The most commonly used PAM sequence recognized by Cas9 protein is 5'-NGG-3 ', where N represents any nucleotide.
If the PAM sequence matches correctly and the gRNA successfully binds to the target site, then Cas9 will cut the DNA double-stranded about 3-4 nucleotides upstream of the PAM sequence .
Alt-R™ CRISPR-Cas9 system- for improving gene editing efficiency
The Alt-R CRISPR-Cas9 system includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPER-Cas9 system has more advantages compared with other methods:
Use Alt-R SpCas9 Nuclease 3NLS for efficient editing
The Alt-R CRISPR-Cas9 system includes the highly efficient Alt-R SpCas9 Nuclease 3NLS. By combining Alt-R SpCas9 Nuclease 3NLS with optimized chemical modification Alt-R CRISPR-Cas9 crRNA and tracrRNA to form an RNP, this system is superior to other editing methods (Figure 1). RNP transfection allows the optimal dose control of the editing complex, and the non-renewable Cas9RNP can be eliminated by endogenous mechanisms in a short time, effectively limiting off-target editing.
Figure 1 Optimized crRNA: tracrRNA is superior to other guide RNA types.
Alt-R™ CRISPR-Cas9 RNA, natural S. pyogenes CRISPR RNA, in vitro transcription (IVT) single-guide RNA (sgRNA) and sgRNA expressed by expression plasmids or gBlocks® gene fragments all target 4 human HPRT loci Points (38087 AS, 38509 S, 38285 AS and 38636 AS). The guide RNA was transfected into HEK-293-Cas9 cells stably expressing Cas9. Mutation detection using the T7EI assay showed that Alt-R CRISPR-Cas9RNA outperformed other guide RNA types at most sites.
Efficient performance simplifies design
The optimized crRNA and tracrRNA in the Alt-R CRISPR-Cas9 system are superior to other CRISPR guide RNA formats (Figure 2). In fact, the system is very efficient, so cr RNA requires minimal design work. The only thing you need to provide is a site-specific 19 or 20 base sequence that is next to the PAM (proximal region sequence adjacent motif; NGG) sequence in your target gene. Using Alt-R CRISPR-Cas9 crRNA, under standard conditions, an average of >80% of the target sequence will show effective editing.
Figure 2 Alt-R™ CRISPR-Cas9 crRNA designed for 92 PAM sites in the STAT3 gene provides high editing efficiency.
Alt-R CRISPR-Cas9 crRNA (n=92), targeting each PAM site in the STAT3 locus. Mutation detection using the T7EI assay showed that 93% of crRNA showed good to excellent performance in HEK-293-Cas9 cells, with an editing efficiency of >20%. Note: T7EI cannot detect single-base deletions or insertions, which will underestimate the editing efficiency.
Some articles published in journals such as Nature and Neuron using IDT Alt-R® CRISPR:
Riddle MR, Aspiras AC, et al. (2018) Insulin resistance in cavefish as an adaptation to a nutrient-limited environment. Nature, 555 : 647–651.
Luo L, Bokil N, et al.. (2017) SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages . Nat Commun, 8 : 14133.
Agudelo D, Duringer A, et al.. (2017) Marker-free coselection for CRISPR-driven genome editing in human cells. Nature Methods, 14 :615–620.
Mikheikin A, Olsen A, et al. (2017) DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle. Nat Commun, 8 : 1665.
Kohler S, Wojcik M, et al.. (2017) Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue . Proc Natl Acad Sci USA, 114 : E4734–E4743.
Seki A, Rutz S. (2018) Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J Exp Med. doi: 10.1084/jem.20171626
Quadros RM, Miura H, et al. (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins . Genome Biology, 18 : 92.
Han X, Liu Z, et al. (2017) Cas9 ribonucleoprotein delivery via microfluidic cell-deformation chip for human T-Cell genome editing and immunotherapy . Adv Biosys, 1 : 1600007.
Andersson M, Turesson H, et al. (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant. doi: 10.1111/ppl.12731
Brinkman EK, Kousholt AN, et al. (2018) Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res. doi: 10.1093/nar/gky164
Kim KW, Tang NH, et al. (2018) A neuronal piRNA pathway inhibits axon regeneration in C. elegans. Neuron, 97 : 1–9.
Al Abdallah Q, Ge W, Fortwendel JR. (2017) A simple and universal system for gene manipulation in Aspergillus fumigatus: in vitro-assembled Cas9 guide RNA ribonucleoproteins coupled with microhomology repair templates. mSphere, 2 : e00446–17.
di Pietro F, Valon L, et al.. (2017) An RNAi screen in a novel model of oriented divisions identifies the actin-capping protein Z β as an essential regulator of spindle orientation. Curr Biol, 27 : 2452–2464.
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