An Off the Shelf Method for Precise Gene Editing in E. coli

Swings, et. al. (2018) Nature Communications 9:2231. https://www.nature.com/articles/s41467-018-04651-5?_ga=2.91952154.1695293922.1528489497-266046807.1528489497

Precise editing with CRISPR through homology directed repair requires careful selection of gRNA sequences and template source.  This can be a challenge as each new site requires the design of new gRNAs.  To streamline this process, Swings et. al. describes a method using the Keio E. coli knockout collection and a gRNA that targets the flanking ends of the inserted kanamycin cassette.  This collection and reagents can be used to alter each gene in the E. coli genome through homology directed repair with the researcher only having to design the homology template.

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.

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 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.

CRISPR Knock Out of FREP1 Suppresses Malaria Infection in Mosquitoes

EurekaAlert! 8 March 2018, https://www.eurekalert.org/pub_releases/2018-03/p-cts030218.php

A new study in PLOS Pathogens demonstrates that CRISPR knockout of FREP1 in Anopheles gambiae mosquitos suppressed Plasmodium parasites infections.  This could potentially be used as a gene drive mechanism to eliminate malaria infections.  The inactivation of FREP1 did result in reduced blood-feeding, lower egg hatching rate, slowed development, and reduced longevity after feeding on blood, raising concerns that the modified mosquito may not be able to compete with its wild-type counterparts.

Modeling CRISPR Hybridization Kinetics

Klein, M. et. al. (2018) Cell Reports 22:1413-1426. https://www.ncbi.nlm.nih.gov/pubmed/29425498

For CRISPR-based therapies to become viable, the off-target effects of the nucleases must be controlled.  To enhance the guide selection, this paper presents a kinetic model using four parameters that can mechanistically explain guide binding and off-target predictions.

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.