Genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese)!

For the paper:

For the plasmids: 


Up, down, and out: optimized libraries for CRISPRa, CRISPRi, and CRISPR-knockout genetic screens

Kendall R SansonRuth E HannaMudra HegdeKatherine F DonovanChristine StrandMeagan E SullenderEmma W VaimbergAmy GoodaleDavid E RootFederica Piccioni, John G Doench


Advances in CRISPR-Cas9 technology have enabled the flexible modulation of gene expression at large scale. In particular, the creation of genome-wide libraries for CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) has allowed gene function to be systematically interrogated. Here, we evaluate numerous CRISPRko libraries and show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we developed genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese). Negative selection screens showed that Dolcetto substantially outperforms existing CRISPRi libraries with fewer sgRNAs per gene and achieves comparable performance to CRISPRko in the detection of gold-standard essential genes. We also conducted positive selection CRISPRa screens and show that Calabrese outperforms the SAM library approach at detecting vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.

Great links for making sense of TCR nomenclature!

Mouse Vb6 is the same as TRBV19.  Human Vb3 is the same as TRBV28. It´s confusing, to say the least.

Here are some links for finding antibodies for different TCR genes!!:

Human TRAV:

Human TRBV:

Mouse TRAV:

Mouse TRBV:

For a bit more of an overview, as suggested to me by Dr. Daniel Pellicci, have a look at “Correspondence between nomenclatures” also at IMGT / 

Interesting study describing the transcriptional response of HSCs to CRISPR from the Porteus lab

Mol Ther. 2018 Jul 10. pii: S1525-0016(18)30261-2. doi: 10.1016/j.ymthe.2018.06.002. [Epub ahead of print]

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+Hematopoietic Stem and Progenitor Cells.


Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.


RNA expression analysis; genome editing; hematopoietic stem cells

PMID: 30005866

“Accurate analysis of genuine CRISPR editing events with ampliCan”

Kornel LabunXiaoge GuoAlejandro ChavezGeorge ChurchJames A GagnonEivind Valen
“We present ampliCan, an analysis tool that unites identification, quantification and visualization of genuine genome editing events from CRISPR amplicon sequencing data. ampliCan overcomes methodological challenges suffered by other tools to estimate the true mutational efficiency in a high-throughput automated fashion. ampliCan controls for biases at every step of the analysis and generates reports that allow users to quickly identify successful editing events or potential issues with their experiments. We benchmarked ampliCan against other leading tools demonstrating that it outperformed all in the face of common confounding factors.”


Am I ready for CRISPR? A user’s guide to genetic screens by Dr. John G. Doench.

Highly recommended review regarding CRISPR screens:


Exciting new technologies are often self-limiting in their rollout, as access to state-of-the-art instrumentation or the need for years of hands-on experience, for better or worse, ensures slow adoption by the community. CRISPR technology, however, presents the opposite dilemma, where the simplicity of the system enabled the parallel development of many applications, improvements and derivatives, and new users are now presented with an almost paralyzing abundance of choices. This Review intends to guide users through the process of applying CRISPR technology to their biological problems of interest, especially in the context of discovering gene function at scale.

PMID: 29199283