CRISPR Knockdown

The technique for obtaining knockdowns with CRISPR entails the use of proteins known as CRISPR-associated genes, which can be programmed to insert exogenous DNA fragments into a CRISPR locus. Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes are reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. CRISPR is commonly used by researchers for obtaining knockdowns in various mouse models, for the purpose of fine-tuning their genetic research.

CRISPR Knockdown Contains The Following Sections

CRISPR-mediated Knockdown Means CRISPR Interference (CRISPRi)

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa). Based on the bacterial genetic immune system-CRISPR (clustered regularly interspaced short palindromic repeats) pathway [1], the technique provides a complementary approach to RNA interference. The difference between CRISPRi and RNAi, though, is that CRISPRi regulates gene expression primarily on the transcriptional level, while RNAi controls genes on the mRNA level.

CRISPRi can sterically repress transcription by blocking either transcriptional initiation or elongation. This is accomplished by designing sgRNA complementary to the promoter or the exonic sequences. dCas9 binds to a tracrRNA:precursor crRNA and recruits RNase III to process the precursor and liberate the crRNA. The crRNA directs binding of dCas9 to promoter or open reading frame regions to prevent RNAP binding or elongation, 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.

A cas9 mutant (dcas9) that abrogate nuclease activity

Fig 1. Plasmid pdCas9 encodes a cas9 mutant containing D10A and H840A substitutions (red asterisks) that abrogate nuclease activity.

It has been suggested that this is due to 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 almost 99.9%; in human cells, up to 90% repression was observed [2]. Several methods have been developed to improve the efficiency of transcriptional modulation. Identification of the transcription start site of a target gene and considering the preferences of sgRNA improves efficiency, as does the presence of accessible chromatin at the target site.

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 99% in human cells [4]. the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

A significant portion of the genome (both reporter and endogenous genes) in eukaryotes has been shown to be targetable using lentiviral constructs to express dCas9 and sgRNAs, with comparable efficiency to existing techniques such as RNAi and TALE proteins. In tandem or as its own system, CRISPRi could be used to achieve the same applications as in RNAi [4]. For bacteria, gene knockdown by CRISPRi has been fully implemented and characterized (off-target analysis, leaky repression) for both Gram-negative E. coli [5,6] and Gram-positive B. subtilis [7].

CRISPR Knockdown Related References

1. Barrangou, R et al. (2007). CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science. 315 (5819): 1709–1712.
2. Qi, L. S et al. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 152 (5): 1173–83.
3. Bikard D et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41:7429–37.
4. Gilbert, L. A et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 154 (2): 442–51.
5. Jiang, W et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology. 31 (3): 233–9.
6. Li, X et al. (2016). tCRISPRi: tunable and reversible, one-step control of gene expression. Scientific Reports. 6: 39096.
7. Peters JM et al. (2016). A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria. Cell. 165 (6): 1493–1506.