GEN Summarizes Findings at the Fourth Annual Precision CRISPR Congress

Katy Liszewski, GEN, 15 April 2018, https://www.genengnews.com/gen-articles/true-crispr-a-genetic-genre-with-novel-twists/6296

Staying on top of new CRISPR developments can be simple as attending conferences. However, as that is not feasible for everyone, GEN has summarized the findings presented at the Precision CRISPR Congress.  This article covers updates in multiplexing, nanocarriers, plant breeding, and T-cell therapies.

Essential Genes Identified in Pluripotent Stem Cells

GenomeWeb, 17 April 2018, https://www.genomeweb.com/cell-biology-research/researchers-identify-essential-genes-pluripotent-stem-cells-through-crispr#.WtpCdMgvzcs

Researchers in Jerusalem, Israel, used haploid human pluripotent stem cells and a CRISPR/Cas screen to identify essential and growth-restriction genes.  The researchers found that most of the essential genes were nuclear and mitochondrial in function.  They hope that publication of this library will reveal key aspects of cellular essentiality.

Bridged Nucleic Acids Improve Cas9 Specificity

Cromwell, CR., et. al. (2018) Nature Communications. 9:1448. https://www.ncbi.nlm.nih.gov/pubmed/29654299

Off-target effects resulting from CRISPR gene editing could hamper the systems adoption as a therapeutic technique.  In a recent Nature Communications publication, Cromwell et. al. demonstrate that use of next-generation bridged nucleic acids (BNA) and locked nucleic acids (LNA) at specifics spots in the crRNA can reduce off-target cleavage by multiple orders of magnitude.  The authors demonstrate that this is due to the modified nucleic acids slowing Cas9 kinetics.

New Tool for Designing Optimal sgRNAs

Dhanjal JK., et. al. (2018) Genomics https://www.ncbi.nlm.nih.gov/pubmed/29605634

The ideal sgRNA will target a single loci in the genome and result in no off-target effects. Identifying such a sequence can involve significant amounts of trial and error.  A new tool coined CRISPcut seeks to aid in this process by predicting sgRNAs for four different types of Cas9 nucleases and different PAM sequences in human cells.  Additionally, experimental data restricts this tool to the open chromatin sequences and can predict sequences for paired nickases.

USA Will Not Regulate CRISPR-Edited Crops

Diana Kwon, 02 April 2018, The Scientist, https://www.the-scientist.com/?articles.view/articleNo/52209/title/USDA-Will-Not-Regulate-CRISPR-Edited-Crops/

In a statement released on March 28 the USDA stated that they will not regulate plants that have been modified through genome editing as long as the same genotype could have been produced through traditional breeding methods.  This announcement has the potential to shave tens of millions of dollars off the cost of crop develop.

Flawed CRISPR Study Retracted

Megan Molteni, Wired Science, 02 April 2018 https://www.wired.com/story/a-flawed-study-shows-how-little-we-understand-crisprs-effects/

CRISPR has shown great promise as a therapeutic, but off-target effects remain a concern.  Last May a small case study determined that CRISPR therapeutics may lead to dangerous levels off off-target mutations, resulting in the shares of CRISPR startups plummeting.  Due to the inability of other scientists to reproduce this work, Nature Methods has retracted the paper.  This episode reveals is a genuine lack of knowledge on how CRISPR acts in cells and how many off-target effects are actually introduced.  Scientists have been working to answer these questions and develop technology to screen for off-target mutations, but to date not enough research has been completed to fully answer these questions.

Automating Cellular Extraction from Live Zebrafish

Lambert, CJ., et. al. (2018) PLoS One 13:e0193180. https://www.ncbi.nlm.nih.gov/pubmed/29543903

Zebrafish have long been a valuable model system with CRISPR gene editing providing even greater opportunities.  One of the major bottlenecks in gene editing is the identification of modified larvae and embryos.  Lambert et. al. developed a zebrafish embryonic genotyping device coined ZEG that uses microfluidic harmonic oscillation of the fish on a glass surface to obtain tissue suitable for genetic analysis.  The researchers determined that this process does not affect body morphology, development, or motor behavior tests.

Streamlining In Vitro Transcription of Cas9 mRNA

Redel, BK., et. al. (2018) Biotechniques 64:118-124. https://www.biotechniques.com/BiotechniquesJournal/2018/March/Single-step-production-of-Cas9-mRNA-for-zygote-injection/biotechniques-366753.html

With the use of DNA-free CRISPR gene editing on the rise, more researchers are turning to in vitro transcription (IVT) of Cas9 mRNA for delivery of the gene editing protein.  Traditionally this involves the addition of a 5’ cap and 3’ polyadenylation post-IVT to ensure transcript stability.  This paper describes a new method that does not require these additional steps by using a new Cas9 mRNA that contains a triple helical tail originating from the mMalat1 gene instead of the traditional polyA tail.  Additionally, this new mRNA has a defined and fixed length that allows researchers to better assesses mRNA degradation.

CRISPR Startup and Salk Institute Both Publish on New Cas13d

Christie Rizk, GenomeWeb, 23 March 2018, https://www.genomeweb.com/gene-silencinggene-editing/crispr-startup-arbor-biotechnologies-salk-institute-concurrently-discover#.WrVUlKjwaUk

Startup Arbor Biotechnologies and the Salk Institute simultaneously published the discovery of a new class 2 enzyme dubbed Cas13d.  This new Cas system is smaller than previous Cas13 proteins and uses a single RNA guide, possibly providing for its potential development into a simpler system than the more well-known Cas9.  More research into specificity and off-target rates are needed before Cas13d is ready for widespread adoption.

CRISPR Based Methods for Annotation of Regulatory DNA

Klann TS, et. al. (2018) Current Opinion in Biotechnology. 52:32-41. https://www.ncbi.nlm.nih.gov/pubmed/29500989

High-throughput screens for coding regions of the genome have long relied on RNAi technology, but techniques for screening the non-coding regions have not existed until recently.  This review covers how CRISPR technology can be harnessed for annotation of the non-coding regions through genomic and epigenomic screens.