Single guide RNA

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sgRNA at Cas9 (green)
Base pairing between the sgRNA and target sequence
Steps in designing an sgRNA

Single Guide RNA (sgRNA) is an artificial RNA that is used in the CRISPR / Cas method , CRISPRi or CRISPRa in combination with Cas9 or Cas12b .

properties

The sgRNA forms a secondary structure called an R-loop . It can be used in bacteria , yeast , fruit flies , zebrafish, and mice . It is then bound by Cas proteins of type I and II. Cas9 naturally binds two RNAs, the crRNA and the tracrRNA , while in the method only one sgRNA consisting of sequences of the two RNAs is used. This means that only one RNA has to be cloned for the CRISPR / Cas method . An sgRNA consists of the 20 nucleotides upstream (in the 5 'direction) of a Protospacer Adjacent Motif (PAM) of the target DNA to be cut and part of the tracrRNA. There is preferably a guanine nucleotide ( GC clamp ) at the 5 'end of the 20 nucleotides (position 1) and an adenine or thymine nucleotide four nucleotides in front of the PAM (position 17) . Programs for identifying 20 nucleotides in front of a PAM or for designing an sgRNA are, for example, CHOPCHOP, CasOFFinder, FlyCRISPR, CRISPR-ERA, SgRNA Designer, CRISPOR, E-CRISP and CRISPRdirect.

In most cases, viral vectors are transduced or plasmids are transfected to introduce the sgRNA into eukaryotic cells . When used for gene therapy in eukaryotes, a eukaryotic promoter is used. If the Cas protein and the sgRNA are introduced into a cell , the sgRNA is generated beforehand by in vitro transcription from a vector, for example with a T7 promoter .

Web links

Individual evidence

  1. ^ A b F. Jiang, DW Taylor, JS Chen, JE Kornfeld, K. Zhou, AJ Thompson, E. Nogales, JA Doudna: Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. In: Science . Volume 351, number 6275, February 2016, pp. 867-871, doi : 10.1126 / science.aad8282 , PMID 26841432 , PMC 5111852 (free full text).
  2. ↑ W. Jiang, D. Bikard, D. Cox, F. Zhang, LA Marraffini: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. In: Nature Biotechnology . Volume 31, number 3, March 2013, pp. 233-239, doi : 10.1038 / nbt.2508 , PMID 23360965 , PMC 3748948 (free full text).
  3. ↑ JM Peters, A. Colavin, H. Shi, TL Czarny, MH Larson, S. Wong, JS Hawkins, CH Lu, BM Koo, E. Marta, AL Shiver, EH Whitehead, JS Weissman, ED Brown, LS Qi, KC Huang, CA Gross: A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria. In: Cell . Volume 165, number 6, June 2016, pp. 1493–1506, doi : 10.1016 / j.cell.2016.05.003 , PMID 27238023 , PMC 4894308 (free full text).
  4. ↑ XT Li, Y. Jun, MJ Erickstad, SD Brown, A. Parks, DL Court, S. Jun: tCRISPRi: tunable and reversible, one-step control of gene expression. In: Scientific Reports . Volume 6, 12 2016, p. 39076, doi : 10.1038 / srep39076 , PMID 27996021 , PMC 5171832 (free full text).
  5. ^ JE DiCarlo, JE Norville, P. Mali, X. Rios, J. Aach, GM Church: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. In: Nucleic acids research. Volume 41, number 7, April 2013, pp. 4336-4343, doi : 10.1093 / nar / gkt135 , PMID 23460208 , PMC 3627607 (free full text).
  6. ↑ GW Thickbroom, FL Mastaglia: Cerebral events preceding- self-paced and visually triggered saccades. A study of presaccadic potentials. In: Electroencephalography and clinical neurophysiology. Volume 62, Number 4, July 1985, pp. 277-289, doi : 10.1016 / 0168-5597 (85) 90005-x , PMID 2408874 .
  7. ↑ WY Hwang, Y. Fu, D. Reyon, ML Maeder, SQ Tsai, JD Sander, RT Peterson, JR Yeh, JK Joung: Efficient genome editing in zebrafish using a CRISPR-Cas system. In: Nature Biotechnology . Volume 31, number 3, March 2013, pp. 227-229, doi : 10.1038 / nbt.2501 , PMID 23360964 , PMC 3686313 (free full text).
  8. ↑ H. Wang, H. Yang, CS Shivalila, MM Dawlaty, AW Cheng, F. Zhang, R. Jaenisch: One-step generation of mice carrying mutations in multiple genes by CRISPR / Cas-mediated genome engineering. In: Cell . Volume 153, number 4, May 2013, pp. 910-918, doi : 10.1016 / j.cell.2013.04.025 , PMID 23643243 , PMC 3969854 (free full text).
  9. ↑ M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, JA Doudna, E. Charpentier: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. In: Science . Volume 337, number 6096, August 2012, pp. 816-821, doi : 10.1126 / science.1225829 , PMID 22745249 , PMC 6286148 (free full text).
  10. ↑ How to design sgRNA sequences. In: takarabio.com. Retrieved February 2, 2019 .
  11. ↑ CHOPCHOP. In: chopchop.cbu.uib.no. Retrieved January 30, 2019 .
  12. ↑ CRISPR RGEN Tools. In: rgenome.net. Retrieved January 30, 2019 .
  13. ^ C. Dustin Rubinstein, Ed O'Connor-Giles, Kate O': CRISPR Optimal Target Finder. In: tools.flycrispr.molbio.wisc.edu. Retrieved January 30, 2019 .
  14. ↑ CRISPR-ERA. In: crispr-era.stanford.edu. Accessed January 30, 2019 .
  15. ↑ sgRNA Designer: CRISPRko. In: portals.broadinstitute.org. Retrieved January 30, 2019 .
  16. ↑ CRISPOR. In: crispor.tefor.net. Accessed January 30, 2019 .
  17. ↑ E-CRISP design. In: e-crisp.org. January 1, 2013, accessed January 30, 2019 (enc).
  18. ^ SE Mohr, Y. Hu, B. Ewen-Campen, BE Housden, R. Viswanatha, N. Perrimon: CRISPR guide RNA design for research applications. In: The FEBS journal. Volume 283, number 17, 09 2016, pp. 3232–3238, doi : 10.1111 / febs.13777 , PMID 27276584 , PMC 5014588 (free full text).