Allele Specific CRISPR Editing

Sharon Begley, 02 February 2018, STAT, https://www.statnews.com/2018/02/02/crispr-blindness-retinitis-pigmentosa/

Many diseases are the result of a single mutated allele.  To correct these with a CRISPR-based system, the mutated allele should be targeted while the healthy allele is left untouched.  In one of the first papers to be published in The CRISPR Journal, researchers have developed a method to target the “broken” allele while leaving the healthy allele untouched.  This proof of concept work was done in a mouse retinitis pigmentosa model, where blindness is caused by a single nucleotide change in one allele.  Results have been published on the BioRxiv prepress server (https://www.biorxiv.org/content/early/2018/01/29/197962).

Increasing Homology Directed Repair Efficiency by “Cold Shock”

Guo, Q., et. al. (2018) Scientific Reports. 8:2080. https://www.ncbi.nlm.nih.gov/pubmed/29391533

Homology directed repair (HDR) has long been plagued by low efficiency, limiting its use in gene editing.  Researchers working with induced pluripotent stem cells (iPSC) have found that by incubating cells at 32°C for 24-48 hours post-transfection, HDR efficiency can be increased by two- to ten-fold.  This type of research could allow for more efficient use of CRISPR HDR.

Epigenome Modifications Create IPSCs

Abby Olena, The Scientist, 25 January 2018, https://www.the-scientist.com/?articles.view/articleNo/51500/title/Stem-Cells-Made-by-Modifying-the-Epigenome-with-CRISPR/

Transitioning somatic cells into induced pluripotent stem cells (IPSCs) has traditionally been a laborious process.  By coupling dCas9 with epigenetic remodeling, researchers were able to activate either Sox2 or Oct4, converting the transfected cells into IPSCs.  The results of this study were published in Cell Stem Cell (https://www.ncbi.nlm.nih.gov/pubmed/29358044).

Could the Immune System Prevent CRISPR Therapies?

Heidi Ledford, Nature, 08 Jan 2018, https://www.nature.com/articles/d41586-018-00335-8

According to research published on the preprint server BioRxiv, 79% of studied blood samples contained antibodies for Staphylococcus aureus Cas9 and 65% of samples contained antibodies for Streptococcus pyogenes Cas9.  Additionally, 46% of the 13 adult participates contained T cells targeting S. aureus Cas9.  While these data have not been peer-reviewed, it demonstrates potential challenges faced by in vivo editing with Cas9.

CRISPR Converts Microbes to Tape Recorders

GEN News Highlights, 27 Nov 2017, https://www.genengnews.com/gen-news-highlights/crisprcas-used-to-create-microscopic-data-recorder/81255201

Scientists have created a CRISPR/Cas system in E. coli that allows for the recording of events and the time at which they occurred.  This was done by having the cell insert CRISPR spacers at regular occurring sets of time, however when a signal was detected the bacteria would insert a signal sequence instead.  This locus can then be read using pre-existing sequencing tools providing a readout of events.  The researchers hope this technology could be used to record biological events.

CRISPR May Not Revolutionize Mitochondrial Genome Editing

Gammage, P.A.  et. al. (2017) Trends in Genetics. https://www.ncbi.nlm.nih.gov/pubmed/29179920

Mitochondrial DNA has increased in prominence in both biology and medicine as a major player for various diseases.  Attempts to alter the genetic information of mitochondria have made significant advances using zinc finger and TALEN nucleases.  These protein only nucleases are easily delivered into the mitochondria through existing machinery.  While CRISPR has revolutionized genome editing, it is struggling in mitochondria due to the lack of an endogenous mechanism for nucleic acid transport into the mitochondria, preventing its adoption.

New Cas9 Cryo-EM Structure Solved

Huai, C. et. al. (2017) Nature Communications. 8:1375. https://www.ncbi.nlm.nih.gov/pubmed/29123204

Researchers have solved a 5.2 Å cryo-EM structure of Cas9 complexed with sgRNA and target DNA.  The structure found that the HNH domain moves closer to the DNA than previously reported and suggests that this new structure resembles a DNA cleavage-activating structure of Cas9.

Identification of a Thermostable Cas9

Harrington L.B. et. al. (2017) Nature Communications 8:1424. https://www.ncbi.nlm.nih.gov/pubmed/29127284

The CRISPR/Cas systems used for genome editing to date have come from mesophilic bacteria, preferring temperatures of 20-45°C, preventing their use at higher temperatures.  Harrington et. al. have identified a Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) that is active in temperature up to 70°C, providing a much wider range of possibilities.  Additionally, GeoCas9 showed greater stability as an RNP complex in human plasma, opening the door to possible therapeutic uses.

Could CRISPR/Cas Claims Inhibit Innovation?

Gray, B.N and Spruill, W.M. (2017) Nature Biotechnology 35:630-633. https://www.ncbi.nlm.nih.gov/pubmed/28700549

The ongoing patent battle between the Broad Institute and the University of California-Berkeley provides difficulties for researchers and companies wishing to develop CRISPR/Cas technology, though this is not the only barrier.  This article describes the broad claims that have been granted or that are being investigated and presents the argument that these claims are overly broad and could limit the genome editing field.

Using Gene Drives to Study Fungal Pathogenicity

GEN News Highlights, 30 October 2017, https://www.genengnews.com/gen-news-highlights/crispr-drives-out-fungal-resistance/81255106

Gene drives have been described as a way to eliminate pests, notably the mosquito, from the environment. However, they are a powerful research tool as well.  Candida albicans can be notoriously difficult to study due to its diploid nature.  By combining a newly discovered haploid C. albicans and CRISPR/Cas gene drive technology, researchers have been able to rapidly create diploid knockouts for study (https://www.ncbi.nlm.nih.gov/pubmed/29062088).  The creation of these mutants could serve to increase the pace of drug discovery to combat this and other fungal pathogens.