CRISPRa

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CRISPRa (from CRISPR activation ) is a biochemical method for generating transcription factors with a determinable DNA target sequence.

properties

Base pairing between the sgRNA and target sequence

Like the CRISPR / Cas method and CRISPRi, CRISPRa is based on an antiviral defense mechanism in bacteria, the CRISPR . CRISPRa 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 . dCas9 is combined for CRISPRa with a transcription factor, thereby creating a fusion protein from dCas9 and transcription factor - either N - or C -terminal . The proportion of the transcription factor binds other proteins that are necessary for gene expression.

The target sequence that is bound in CRISPRa requires a Protospacer adjacent motif for dCas9 to bind. In addition, the sgRNA must have a sequence that is reverse-complementary to the target sequence, so that binding by base pairing between sgRNA and target sequence can take place. Furthermore, it should be ensured that the sgRNA only binds to the desired target sequence and not to other identical or very similar target sequences in the organism used. The target sequence is often placed in a promoter area in order to bring the transcription factor close to a gene. Occasionally, different target sequences and transcription factors are used simultaneously to enhance gene expression.

CRISPRa is used for targeted gene activation, such as altering cell differentiation (including creating induced pluripotent stem cells ), high-throughput gene screening, and protein overexpression . The sgRNA and the fusion protein (from dCas9 and transcription factor) are encoded by a plasmid or a viral vector and introduced into cells. By using these two variants in combination with an inducible promoter, gene expression can be timed.

Types

In eukaryotes

In eukaryotes , more precisely in cell cultures of fruit flies , house mice and humans, the transcription factors used are P64-p65-Rta, SAM ( Synergistic Activation Mediator) and SunTag. The target sequence should be 50 to 500 nucleotides in front of the gene to be activated.

VP64-p65-Rta

dCas9-VP64-p65-Rta

The VP64-p65-Rta (also VPR ) uses dCas9 and sgRNA in connection with the three transcription factors Vp64, p65 and Rta in series at the C terminus of dCas9. By using three transcription factors, gene expression is increased compared to just one. By using different sgRNA, several target sequences and thus also different genes can be activated within a cell.

Synergistic activation mediator

dCas-SAM

The Synergistic activation mediator (SAM) uses the three transcription factors MS2, p65 and HSF1. In the sgRNA used for this, there are 2 aptamers in the tetra loop and the stem loop for binding MS2, so that MS2 dimers are formed. The MS2 is a fusion protein with p65 and HSF1.

Sun day

Sun day

The SunTag is a repetitive epitope (10-fold) that has been fused to a modified dCas9 and binds several scFv fragments with the transcription factor VP-64. In order for the scFv fragments to be transported into the cell nucleus , they are modified with a nuclear localization signal .

In prokaryotes

In prokaryotes , CRISPRa variants are used, which initiate gene expression on σ 70 - or σ 54 -dependent promoters. When using σ 54 -dependent promoters as the target sequence, an up to 1000-fold increase in gene expression and a 70-fold difference in correct binding compared to base mismatching of the binding to the target sequence have been described.

literature

Individual evidence

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