Author: fredrikwermeling

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

For the paper: https://www.biorxiv.org/content/early/2018/07/02/356626

For the plasmids: https://www.addgene.org/browse/article/25689/ 

 

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

Kendall R Sanson, Ruth E Hanna, Mudra Hegde, Katherine F Donovan, Christine Strand, Meagan E Sullender, Emma W Vaimberg, Amy Goodale, David E Root, Federica Piccioni, John G Doench
doi: https://doi.org/10.1101/356626

Abstract

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:

http://www.imgt.org/IMGTrepertoire/Regulation/antibodies/human/TRA/TRAV/Hu_TRAVMab.html

Human TRBV:

http://www.imgt.org/IMGTrepertoire/Regulation/antibodies/human/TRB/TRBV/Hu_TRBVMab.html

Mouse TRAV:

http://www.imgt.org/IMGTrepertoire/index.php?section=Regulation&repertoire=antibodies&species=mouse&group=TRAV

Mouse TRBV:

http://www.imgt.org/IMGTrepertoire/index.php?section=Regulation&repertoire=antibodies&species=mouse&group=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 /  http://www.imgt.org/IMGTrepertoire/LocusGenes/#J 

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.

Cromer MK1, Vaidyanathan S1, Ryan DE2, Curry B2, Lucas AB2, Camarena J1, Kaushik M1, Hay SR1, Martin RM1, Steinfeld I2, Bak RO1, Dever DP1, Hendel A3, Bruhn L2, Porteus MH4.

Abstract

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.

KEYWORDS:

RNA expression analysis; genome editing; hematopoietic stem cells

PMID: 30005866

“Accurate analysis of genuine CRISPR editing events with ampliCan”

Kornel Labun, Xiaoge Guo, Alejandro Chavez, George Church, James A Gagnon, Eivind Valen
doi: https://doi.org/10.1101/249474
“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:

https://www.nature.com/articles/nrg.2017.97.pdf

Abstract

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

iBiology – A fantastic series of biology videos. Highly recommended!

https://www.ibiology.org/biology-topics/

“iBiology’s mission is to bring the stories of cutting-edge research in the life sciences to a global audience without paywalls or other barriers to learning. Our primary means of communication is through videos, so the scientists themselves explain their research and the process of scientific discovery.

By providing a direct link to scientists, iBiology bridges the divides that have emerged between the lab bench and the classroom, and academia and the general public. We also provide special materials geared for educators and professional development for science trainees. While not all the content will be accessible to every audience level, all of our products are infused with the same spirit: that scientific progress is fueled by an open exchange of ideas. Thus this project reflects the ethos of science, as hundreds of researchers have donated their time and effort to share their insights and the excitement of discovery on this open forum.

iBiology.org was founded in 2006 by Ron Vale, a professor at University of California, San Francisco and a Howard Hughes Medical Institute Investigator. It has grown to include a wide variety of materials that now consists of hundreds of talks, lectures, interviews, courses, documentary storytelling, and other materials.”