CRISPRi

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CRISPRi-mediated inhibition of initiation or elongation

CRISPRi (from CRISPR interference ) is a biochemical method to inhibit a specific transcription that inhibits gene expression of the blocked gene ( gene knockdown ).

properties

Like the CRISPR / Cas method and CRISPRa, CRISPRi is based on an antiviral defense mechanism in bacteria, the CRISPR . CRISPRi uses an RNA - protein complex , which can bind to a DNA sequence, where a gene expression of desired genes may initiate. The property of Cas9 is used with an sgRNA to bind to a desired DNA sequence. Since Cas9 and sgRNA naturally also cut the bound DNA, a mutant of Cas9 called dCas9 (from dead Cas9 ) is used, which can no longer cut DNA because the endonuclease function for both DNA strands through certain point mutations ( D 10 A and H 840A ) is deactivated. In eukaryotic cells, dCas9 is used with a nuclear localization signal so that dCas9 is imported into the nucleus . In bacteria, CRISPRi can be used in both Gram-negative E. coli and Gram-positive B. subtilis . Through bacterial conjugation , CRISPRi can be passed on to other bacteria and bacterial species in the sense of a horizontal gene transfer . Due to the simple modular design of the sgRNA, CRISPRi is suitable for high throughput screening . By using several sgRNAs, several genes can be inhibited in parallel.

To increase the inhibitory effect, a fusion protein of dCas9 with a repressor can be used, e.g. B. with the protein domain Krüppel associated box (KRAB), whereby the transcription of the bound gene in human cells is suppressed to 99%. Other factors that influence the efficiency of CRISPRi are the binding of the correct sequence of the starting point of transcription, the design of the sgRNA and accessible areas in the chromatin . Suboptimal binding of the sgRNA to the target DNA sequence can also result in lower inhibition rates.

The need for a Protospacer Adjacent Motif (PAM) to be present in the DNA to be bound limits the number of possible target sequences. Chromatin folds and DNA modifications could disrupt the binding of the dCas9-sgRNA complex. CRISPRi can also inhibit other genes in the immediate vicinity of the target sequence that are, for example, spliced or lie on the opposite DNA strand. A bidirectional promoter can also be inhibited in both directions.

An alternative method of inhibiting gene expression uses RNA interference . While CRISPRi inhibits the transcription of a particular gene, RNA interference leads to the degradation of certain RNA sequences.

Individual evidence

  1. ↑ LS Qi, MH Larson, LA Gilbert, JA Doudna , JS Weissman , AP Arkin, WA Lim: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. In: Cell . Volume 152, number 5, February 2013, pp. 1173-1183, doi : 10.1016 / j.cell.2013.02.022 , PMID 23452860 , PMC 3664290 (free full text).
  2. ↑ R. Barrangou , C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, DA Romero, P. Horvath : CRISPR Provides acquired resistance against viruses in prokaryotes. In: Science . Volume 315, Number 5819, March 2007, pp. 1709-1712, doi : 10.1126 / science.1138140 , PMID 17379808 .
  3. ↑ 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).
  4. ↑ DJ Brocken, M. Tark-Dame, RT Dame: dCas9: A Versatile Tool for Epigenome Editing. In: Current issues in molecular biology. Volume 26, 2018, pp. 15-32, doi : 10.21775 / cimb.026.015 , PMID 28879853 .
  5. ↑ LS Qi, MH Larson, LA Gilbert, JA Doudna, JS Weissman, AP Arkin, WA Lim: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. In: Cell . Volume 152, number 5, February 2013, pp. 1173-1183, doi : 10.1016 / j.cell.2013.02.022 , PMID 23452860 , PMC 3664290 (free full text).
  6. ↑ 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).
  7. ↑ 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).
  8. ↑ 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).
  9. ↑ W. Ji, D. Lee, E. Wong, P. Dadlani, D. Dinh, V. Huang, K. Kearns, S. Teng, S. Chen, J. Haliburton, G. Heimberg, B. Heineike, A Ramasubramanian, T. Stevens, KJ Helmke, V. Zepeda, LS Qi, WA Lim: Specific gene repression by CRISPRi system transferred through bacterial conjugation. In: ACS synthetic biology. Volume 3, number 12, December 2014, pp. 929-931, doi : 10.1021 / sb500036q , PMID 25409531 , PMC 4277763 (free full text).
  10. ↑ a b c d M. H. Larson, LA Gilbert, X. Wang, WA Lim, JS Weissman, LS Qi: CRISPR interference (CRISPRi) for sequence-specific control of gene expression. In: Nature protocols. Volume 8, number 11, November 2013, pp. 2180-2196, doi : 10.1038 / nprot.2013.132 , PMID 24136345 , PMC 3922765 (free full text).
  11. ^ LA Gilbert, MH Larson, L. Morsut, Z. Liu, GA Brar, SE Torres, N. Stern-Ginossar, O. Brandman, EH Whitehead, JA Doudna, WA Lim, JS Weissman, LS Qi: CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. In: Cell . Volume 154, number 2, July 2013, pp. 442-451, doi : 10.1016 / j.cell.2013.06.044 , PMID 23849981 , PMC 3770145 (free full text).
  12. ↑ A. Radzisheuskaya, D. Shlyueva, I. Müller, K. Helin: Optimizing sgRNA position markedly Improves the efficiency of CRISPR / dCas9-mediated transcriptional repression. In: Nucleic acids research. Volume 44, number 18, October 2016, p. E141, doi : 10.1093 / nar / gkw583 , PMID 27353328 , PMC 5062975 (free full text).
  13. ↑ Ashish Goyal, Ksenia Myacheva, Matthias Groß, Marcel Klingenberg, Berta Duran Arqué, Sven Diederichs: Challenges of CRISPR / Cas9 applications for long non-coding RNA genes. In: Nucleic Acids Research. , P. E12, doi : 10.1093 / nar / gkw883 . PMID 27694625 . PMC 5388423 (free full text).