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Transcriptional repression via steric hindrance

CRISPR interference (CRISPRi) is a genetic technique which allows for sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway[1].

Background[edit]

The adaptive immune systems of bacteria and archaea consist of CRISPR RNA and CRISPR-associated (cas) genes. This minimal CRISPR system has been successfully adapted for generating gene knockouts in many model organisms (bacteria[2], yeast[3], fruit flies [4], zebrafish [5], mice [6], humans [7]). A complementary technology uses catalytically dead Cas9 (dCas9) lacking endonuclease activity to regulate genes in an RNA-guided manner8. Targeting specificity is determined by complementary base-pairing of a single guide RNA (sgRNA) to the genomic loci. sgRNA is a chimeric noncoding RNA that can be subdivided into three regions: the 20-25 nt base-pairing sequence, a 42 nt dCas9-binding hairpin and a 40 nt terminator[8].

When designing a synthetic sgRNA, only the 20 nt base-pairing sequence is modified from the overall template. Additionally, secondary variables must be considered: off-target effects (for which a simple BLAST run of the base-pairing sequence is required), maintenance of the dCas9-binding hairpin structure and to ensure no restriction sites are present in the new sgRNA as this may pose a problem in downstream cloning steps. Due to the simplicity of sgRNA design, this technology is amenable to genome-wide scaling[9]. CRISPRi relies on the generation of catalytically inactive Cas9. This is accomplished by introducing point mutations in the two catalytic residues (D10A and H840A) of the gene encoding Cas9[8] . In doing so, dCas9 is unable to cleave dsDNA but retains the ability to target DNA. Taken together sgRNA and dCas9 provide a minimum system for gene-specific regulation in any organism[10].

Transcriptional Regulation[edit]

Although the term CRISPRi was initially coined to describe transcriptional interference, it can also be used to describe transcriptional activation and repression[11].

Transcriptional regulation via effector domain

Repression[edit]

CRISPRi can sterically repress transcription in two ways – by blocking transcriptional initiation or elongation. This is accomplished by designing sgRNA complementary to the promoter or exonic sequences, respectively. The level of transcriptional repression for exonic sequences is strand-specific. sgRNA complementary to the non-template strand more strongly represses transcription compared to sgRNA complementary to the template strand. One hypothesis to explain this effect is from the activity of helicase, which unwinds the RNA:DNA heteroduplex ahead of RNA pol II when the sgRNA is complementary to exons of the template strand. In prokaryotes, this steric inhibition can repress transcription of the target gene by 99.7%. Whereas in human cells, up to 63% repression was observed [10].

CRISPRi can also repress transcription via an effector domain. Fusing a repressor domain to dCas9 allows transcription to be further repressed by inducing heterochromatinization. For example, the well-studied Krüppel associated box (KRAB) domain can be fused to dCas9 to repress transcription of the target gene up to 93% in human cells[11].

Activation[edit]

CRISPRi can be used to activate transcription of the target gene by fusing a transcriptional activator to dCas9. For example, the transcriptional activator VP16 can increase gene expression by up to 25-fold in human cells[11].

Applications[edit]

CRISPRi construction workflow

Allelic Series[edit]

Differential gene expression can be achieved by modifying the efficiency of sgRNA base-pairing to the target loci[9]. In theory, modulating this efficiency can be used to create an allelic series for any given gene, in essence creating a collection of hypo- and hypermorphs. These powerful collections can be used to probe any genetic investigation. For hypomorphs, this allows the incremental reduction of gene function as opposed to the binary nature of gene knockouts and the unpredictability of knockdowns. For hypermorphs, this is in contrast to the conventional method of cloning the gene of interest under promoters with variable strength.

Loci Imaging[edit]

Fusing a fluorescent protein to dCas9 allows for imaging of genomic loci in living human cells[12]. This can be used to study chromatin architecture and nuclear organization dynamics.

Stem Cells[edit]

Activation of Yamanaka factors by CRISPRi has been used to induce pluripotency in human and mouse cells providing an alternative method to iPS technology. [13][14]

Limitations[edit]

1.The requirement of a Protospacer Adjacent Motif (PAM) sequence limits the number of potential target sequences. Cas9 and its homologues may use different PAM sequences, and therefore could theoretically be utilized to expand the number of potential target sequences[9].

2.Sequence specificity to target loci is only 14 nt long, which can recur around ~11 times in a human genome[9].

3.Endogenous chromatin modifications may prevent the sequence specific binding of dCas9 and the sgRNA[9].


References[edit]

  1. ^ Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. (2007). "CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes". Science. 315 (5819): 1709–1712. doi:10.1126/science.1138140. PMID 17379808.
  2. ^ Jiang, W; Bikard, D; Cox, D; Zhang, F; Marraffini, L. A. (2013). "RNA-guided editing of bacterial genomes using CRISPR-Cas systems". Nature Biotechnology. 31 (3): 233–9. doi:10.1038/nbt.2508. PMC 3748948. PMID 23360965.
  3. ^ Dicarlo, J. E.; Norville, J. E.; Mali, P; Rios, X; Aach, J; Church, G. M. (2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research. 41 (7): 4336–43. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.
  4. ^ Gratz, S. J.; O'Connor-Giles, K. M. (2013). "Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease". Genetics. 194 (4): 1029–35. doi:10.1534/genetics.113.152710. PMID 23709638.
  5. ^ Hwang, W. Y.; Fu, Y; Reyon, D; Maeder, M. L.; Tsai, S. Q.; Sander, J. D.; Peterson, R. T.; Yeh, J. R.; Joung, J. K. (2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nature Biotechnology. 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.
  6. ^ Wang, H.; Yang, H.; Shivalila, C. S.; Dawlaty, M. M.; Cheng, A. W.; Zhang, F.; Jaenisch, R. (2013). "One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering". Cell. 153 (4): 910–918. doi:10.1016/j.cell.2013.04.025. PMID 23643243.
  7. ^ Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; Dicarlo, J. E.; Norville, J. E.; Church, G. M. (2013). "RNA-Guided Human Genome Engineering via Cas9". Science. 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.
  8. ^ a b Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. (2012). "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity". Science. 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.
  9. ^ a b c d e Larson, M. H.; Gilbert, L. A.; Wang, X; Lim, W. A.; Weissman, J. S.; Qi, L. S. (2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". Nature Protocols. 8 (11): 2180–96. doi:10.1038/nprot.2013.132. PMID 24136345.
  10. ^ a b Qi, L. S.; Larson, M. H.; Gilbert, L. A.; Doudna, J. A.; Weissman, J. S.; Arkin, A. P.; Lim, W. A. (2013). "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression". Cell. 152 (5): 1173–83. doi:10.1016/j.cell.2013.02.022. PMC 3664290. PMID 23452860.
  11. ^ a b c Gilbert, L. A.; Larson, M. H.; Morsut, L; Liu, Z; Brar, G. A.; Torres, S. E.; Stern-Ginossar, N; Brandman, O; Whitehead, E. H.; Doudna, J. A.; Lim, W. A.; Weissman, J. S.; Qi, L. S. (2013). "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes". Cell. 154 (2): 442–51. doi:10.1016/j.cell.2013.06.044. PMC 3770145. PMID 23849981.
  12. ^ Chen, B; Gilbert, L. A.; Cimini, B. A.; Schnitzbauer, J; Zhang, W; Li, G. W.; Park, J; Blackburn, E. H.; Weissman, J. S.; Qi, L. S.; Huang, B (2013). "Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system". Cell. 155 (7): 1479–91. doi:10.1016/j.cell.2013.12.001. PMC 3918502. PMID 24360272.
  13. ^ Kearns, N. A.; Genga, R. M.; Enuameh, M. S.; Garber, M; Wolfe, S. A.; Maehr, R (2014). "Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells". Development. 141 (1): 219–23. doi:10.1242/dev.103341. PMC 3865759. PMID 24346702.
  14. ^ Hu, J; Lei, Y; Wong, W. K.; Liu, S; Lee, K. C.; He, X; You, W; Zhou, R; Guo, J. T.; Chen, X; Peng, X; Sun, H; Huang, H; Zhao, H; Feng, B (2014). "Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors". Nucleic Acids Research. 42 (7): 4375–90. doi:10.1093/nar/gku109. PMC 3985678. PMID 24500196.
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