Eliminating the CRISPR/Cas9 machinery for edited cells

Wang, Y., et. al. Scientific Reports (2016) 6:31145 http://www.ncbi.nlm.nih.gov/pubmed/27503169

Two of the challenges facing CRISPR/Cas9 gene editing are off-target effects and Cas9 toxicity due to prolonged Cas9/gRNA expression.  In order to prevent these adverse outcomes Wang et al. have developed a CRISPR/Cas9 system that will spontaneously eliminate itself co-transforming with a deletion construct that knocks out Cas9 expression during induced crossing over events.

An Agrobacterium based CRISPR gene editing method for Maize

Char, S. N., et. al. Plant Biotechnology Journal, (2016) http://www.ncbi.nlm.nih.gov/pubmed/27510362

While the CRISPR/Cas9 gene editing system has been shown to work in a variety of cell types, including plants and animals, differences in plant biology require methods optimized for individual species.  Using a binary-vector, Agrobacterium delivery system Char et al. where able to achieve mutation frequencies greater than 70%.  To decrease the cost of maize transformation Char et al. demonstrate that it is also possible to mix two Agrobacterium strains to create transgenic plants independently mutated in each target.

Legal costs exceed $15 million in CRISPR patent dispute

Sharon Begley, 12 August 2016, STAT News, https://www.statnews.com/2016/08/12/crispr-patent-fight-legal-bills-soaring/

Editas Medicine has spent $15.6 million in legal fees in defense of the CRISPR patents jointly owned by the Broad Institute and Harvard.  Given that a decision is not expected until 2017, the costs will come in well over the $10 million each side was expected to spend.

CRISPR based conversion of fibroblasts to neuronal cells

Black et al., Cell Stem Cell (2016) http://www.cell.com/cell-stem-cell/pdfExtended/S1934-5909(16)30196-5

Conversion of somatic cells into induced pluripotent stem cells (IPCs) has enabled the creation of model systems that more closely mimic their natural counterparts.  The creation of IPCs cell lines has traditionally involved the overexpression of transgenes which encode specific transcription factors. While effective, this process has been labor intensive and slow.  In this publication Black et al. demonstrate that dCas9 can be used to activate endogenous transcription factors eliminating the need for transgenes.  This procedure has the potential to greatly increase the speed at which IPCs cell lines are created.

Optimizing CRISPR/Cas9 target specificity

Tycko, J., et. al. Molecular Cell (2016) 63:355-370. http://www.ncbi.nlm.nih.gov/pubmed/27494557

Since its discovery in 2012, CRISPR/Cas9 gene editing techniques and procedures have improved rapidly. Despite the rapid improvement, certain aspects of gene editing remain problematic.  Off-target cleavage remains one of the most challenging problems especially in terms of gene editing’s therapeutic potential.  Many different methodologies have been developed to screen for off-target effects, each with strengths and weaknesses.  Tycko et al. review these techniques while also calling for standardized off-target detection methods.

Scientists have trouble replicating NgAgo gene editing

David Cyranoski, Nature News, 8 August 2016, http://www.nature.com/news/replications-ridicule-and-a-recluse-the-controversy-over-ngago-gene-editing-intensifies-1.20387

While the CRISPR/Cas9 system has democratized and streamlined the gene editing process, scientists are always looking for the next big thing.  In May of 2016 the gene editing community thought the next generation of gene editing tools may have been discovered with Gao et. al.’s Nature Biotechnology report on the DNA guided DNA targeting Argonaute protein isolated from Natronobacterium gregoryi (http://www.nature.com/nbt/journal/v34/n7/full/nbt.3547.html). However, few have been able to repeat the work.  In an online survey conducted by the MRC Centre for Regenerative Medicine in the UK only 9 of 106 scientists reported success with the NgAgo gene editing system and Nature Biotechnology has begun an investigation into the report.  The lack of reproducibility may simply be due to lack of details.  Therefore, the authors released a detailed protocol to Addgene.

CRISPR gene drives could be used to modify sex ratios of mosquitos

Galizi, R., et. al. Scientific Reports (2016) 6:31139 http://www.ncbi.nlm.nih.gov/pubmed/27484623

Historically, mosquito populations have been managed in one of two ways, habitat elimination and the application of pesticides.  Recently gene drives have been proposed as a way to eliminate vector borne diseases such as malaria and zika.  These gene drives would use the CRISPR/Cas system to either reduce mosquito populations through sterilization or provide resistance to the pathogen.  Galizi et al. have proposed a new mechanism for gene drive based mosquito elimination by using the CRISPR/Cas system to “shred” the X-chromosome during male meiosis thereby producing a bias towards male offspring in the next generation.  This male bias would decrease the number of breeding females, ultimately resulting in a population crash.

CRISPR editing without the double stranded break

Anna Azvolinsky, The Scientist, 4 August 2014, http://www.the-scientist.com/?articles.view/articleNo/46724/title/CRISPR–No-Cutting-Required/

Researches out of Japan have modified the CRISPR/Cas9 system to contain a deaminase enzyme, allowing Cas9 to modify single base pairs without introducing a double stranded break.  Double stranded breaks result in deleterious NHEJ mutations and are toxic to cells, allowing this new method to potentially introduce mutations more efficiently.  The deaminase was attached to either dead Cas9 or to a “nickase” version.  The deaminase substitutes a uracil for cytosine which is replaced with a thymine in subsequent rounds of DNA replication.  This new tool can modify a cytosine within a 3-5 base pair window of the target site and works in mammalian cell lines.  The team is now working on developing Cas9 systems capable of making any of the four nucleotide substitution combinations.

Could Vibrio natriegens replace E. Coli as the laboratory cloning workhorse?

Chris Palmer, BioTechniques, 3 August 2016 http://www.biotechniques.com/news/biotechniquesNews/biotechniques-364908.html#.V6ScsdIrLct

Since its widespread adoption in the early 1980s, recombinant DNA technology has relied on E. coli.  The lab of George Church has now sequenced and proposed Vibrio natriegens as a better organism for routine cloning.  V. natriegens is a free living nonpathogenic bacteria that has a generation time of 14.8 minutes (2.1x faster than E. coli) reducing wait time for researchers.  Church’s lab is currently working on using CRISPR to help understand V. natriegens’ growth rate.

Creation of Non-Mosaic CRISPR Edited Mice

Hashimoto, M., et al.  Developmental Biology (2016) http://www.ncbi.nlm.nih.gov/pubmed/27474397

Gene editing of mice has traditionally been completed using electroporation or microinjection of the CRISPR machinery into zygotes. However, this can lead to a genetic mosaic where different cells contain different mutations.  The nature of these models hinders the downstream phenotype analysis.  To overcome this Hashimoto et. al. have developed a method to electroporate fertilized zygotes in vitro before the first round of DNA replication, allowing the creation of a non-mosaic edited mouse model.