CRISPR Digest

Since I last wrote about the current CRISPR Craze, the new editions of Nature, Science and Cell have all featured new CRISPR innovations and advances, which I will outline here in an attempt to give a glimpse into the latest research in the genome-engineering field.

For a good general introduction, including both the discovery and recent applications of the CRISPR/Cas9 system, I recommend the review by Hsu et al. (2014) published early this summer.

One of the applications discussed in this review is the so-called CRISPR interference (CRISPRi, in the style of RNA interference/RNAi) method. It was first developed by Qi et al. (2013) and relies on a catalytically inactive Cas9 nuclease (termed dCas9). dCas9 is targeted to a gene of interest using a guide RNA (gRNA), just like in the original mechanism, except that here the dCas9 cannot cleave the double-stranded DNA, but instead physically blocks the RNA polymerase from transcribing the gene, thus reducing/abolishing the expression of the target gene. One of the advantages of this method is that dCas9 expression is inducible, enabling the system to act as an on/off switch for the gene of interest. Furthermore, dCas9 can also be fused to transcriptional activators and this serves to turn on/overexpress genes of interest.

Due to the high efficiency and specificity of the system [beware, however, that of course the system also has off-target effects; see, for example, Lin et al. (2014)]  it has also been utilised for genome-scale CRISPR/Cas9 knockout (GeCKO) screening experiments. In the experiments described by Shalem et al. (2014), CRISPR is used to knock out almost every single gene in the human genome individually. They did this in a melanoma cell line, which harbours the BRAF V600E mutation, and which is therefore sensitive to the BRAF kinase inhibitor vemurafenib: after treatment with the drug most cells died as expected, but those that survived (i.e. had become resistant to vemurafenib) were analysed. Among the survivors  they found gRNAs targeting genes whose loss had previously been implicated in resistance, but they also found genes that had not been linked to resistance yet. Genome-wide screens like this can thus provide the starting points for more detailed, mechanistic studies into various biological processes, including drug resistance mechanisms.

Even more recently Feng Zhang’s group at MIT reported (Platt et al. (2014)) an engineered mouse line expressing the Cas9 nuclease specifically in different tissues (again beware of “specific”: tissue-specific promoters are rarely exclusive to a single tissue type). These mice were used for ex vivo manipulation of dendritic cells (cells of the immune system): after bone marrow cells were harvested, the dendritic cells (which already express Cas9) were infected with a (lenti)virus carrying the gRNA and such edited cells could then theoretically be reintroduced into the mice. They also showed that in vivo experiments can be performed with these mice when the virus expressing the gRNA is directly injected into the brain, for example, or used to make multiple changes in the genomes of lung cells to model cancer initiation/progression.

And only yesterday a paper was published in Nature by O’Connell et al. (2014), showing that CRISPR can also be used for RNA recognition and cleavage, as opposed to DNA cleavage. This can be achieved when the so-called protospacer adjacent motif (PAM), which is required for the recruitment and activation of  the Cas9 nuclease, is provided alongside the gRNA in the form of a short DNA sequence. It may now become feasible to use CRISPR to complement RNAi approaches. The possibilities are endless.

References:

Hsu PD, Lander ES, Zhang F (2014) Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157: 1262-1278

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature advance online publication

Platt Randall J, Chen S, Zhou Y, Yim Michael J, Swiech L, Kempton Hannah R, Dahlman James E, Parnas O, Eisenhaure Thomas M, Jovanovic M, Graham Daniel B, Jhunjhunwala S, Heidenreich M, Xavier Ramnik J, Langer R, Anderson Daniel G, Hacohen N, Regev A, Feng G, Sharp Phillip A, Zhang F (2014) CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell – in press

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152: 1173-1183

Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science 343: 84-87

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