Hämeenlinna

Kaivokatu 16
13100 Hämeenlinna
Y-tunnus: 0499964-0

Puh. +358 3 615 370 Fax. +358 3 682 2039

Espoo

Sinimäentie 10 C
02630 Espoo

Oulu - Huollon toimipiste

Aapistie 7 B
90220 Oulu

Tilaukset, tuotetiedustelut sekä muut tiedustelut: info@immunodiagnostic.fi

Ota yhtettä Tekninen tuki ja huolto
Siirry sisältöön

Four points to consider when planning your CRISPRa studies

CRISPR activation (CRISPRa): A TOOL TO SWITCH GENES ON

In addition to gene editing studies, Streptococcus pyogenes CRISPR-Cas9 system can be utilized also in gene upregulation studies by using a deactivated Cas9 enzyme, dCas9 (dead Cas9). dCas9 retains the ability to bind DNA but lacks nuclease activity and thus does not cut DNA. dCas9 has been engineered to eliminate the nuclease activity by point mutations in the RuvCI and HNH nuclease domains (D10A and H840A) and to boost gene expression by adding transcription activation domains e.g. VP64, p65 and Rta19 (referred to as VPR). CRISPRa dCAs9-VPR complex binds promoter regions and activates transcription of the target gene.

CRISPRa is a neat tool

CRISPRa is a neat tool for researchers to design artificial transcription factors and to see how cells react to upregulation of specific target gene(s) in endogenous context. It can be used to activate large transcripts; for which ORF constructs are difficult to obtain; and different splice variants. It can also be applied in systematic overexpression studies of noncoding genes. Moreover, generation and maintaining of guide RNA screening collections is easier with CRISPRa than with ORFs and both arrayed and pooled screening formats can be used with this system.

 

Guide RNA is the molecule that directs the CRISPRa complex to the target site

Guide RNA can be either synthetic crRNA complexed with a trans-activating tracrRNA or a single guide RNA (sgRNA), where the crRNA has been attached to the tracrRNA. CRISPRa guide RNAs need to attach close to the gene’s promoter region or the transcriptional start site (TSS) to result in gene upregulation. Designing good gRNAs can be challenging because: 1) TSSs are not always well annotated, 2) TSSs may be inaccessible due to presence of other protein factors and 3) cryptic or alternative promoters may be utilized. Using designs based on published guide RNA design tools is strongly recommended as it will increase the likelihood of effective guide RNAs.

Four points to consider when planning your CRISPRa studies:

1. Start by designing workflow for your transcription experiments: generate stable cells that express dCas9-VPR, add guide RNA and perform the phenotypic analysis of gene activation (RT-qPCR, western blotting or immunofluorescence analysis). For longer experiments (more than 96 hours) lentiviral sgRNA is recommended. For short-term experiments (less than 96 hours) synthetic crRNA:tracrRNA or plasmid sgRNA work well.

2. Pooling of synthetic CRISPRa crRNAs can increase transcriptional activation: a crRNA pool achieves equal or better activation than the most functional single crRNA.

3. Multiplexing of crRNAs to more than one gene target with crRNA pools can further increase activation of the target gene: no loss in activation is observed when compared to individual targeting.

4. Transcription activation level depends on the endogenous gene expression level. Thus, when a gene has low levels of expression, high levels of gene activation (100-10,000-fold) can be observed. Vice versa, when gene expression level is already high, further activation is usually less than 100-fold. Therefore, it is important to determine the basal expression level for your cell line of interest to set expectations on the activation you can achieve.

References:

L. A. Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 154, 442–451 (2013).
A. W. Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).
L. A. Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 159, 647–661 (2014).
M. E. Tanenbaum, L. A. Gilbert, L. S. Qi, J. S. Weissman, R. D. Vale, A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 159, 635–646 (2014).
S. Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517, 583–588 (2015).
A. Chavez et al., Highly efficient Cas9-mediated transcriptional programming. Nat. Methods. 12, 326–328 (2015).
J. G. Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 160, 339–350 (2015).
M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).

Read more about CRISPRa here

Read more about CRISPR-Cas in Finnish here

Edit-R predesigned CRISPRa guide RNAs from Dharmacon/Horizon Discovery

Edit-R dCas9-VPR reagent from Dharmacon/Horizon Discovery

You can order your Dharmacon/Horizon Discovery products through Immuno Diagnostic Oy by e-mailing your order to: info@immunodiagnostic.fi

 

Blogin kirjoittaja

Katja-Riikka Louhi, Ph.D.

Tuotepäällikkö, Life Science +358 40 525 5413