Streamlining In Vitro Transcription of Cas9 mRNA

Redel, BK., et. al. (2018) Biotechniques 64:118-124.

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,

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.

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.

Researchers Unable to Reproduce CRISPR Off-Target Problems

Kristen V Brown, Gizmodo, 05 March 2018,

In 2017, a publication raised the alarm over potential off-target effects during CRISPR therapies.  Multiple research groups set out to reproduce the study and found that the off-target effects initially reported were over-stated.  These new studies have worked to restore confidence that the CRISPR gene editing technology may be suitable for gene therapies.

CRISPR Knock Out of FREP1 Suppresses Malaria Infection in Mosquitoes

EurekaAlert! 8 March 2018,

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.

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.

Pig Organs Could Provide Relief to Those in Organ Failure

Alice Park, Time Magazine, 15 February 2018,

Due to limited amounts of viable donated organs, thousands of people die each year in the US waiting for a transplant.  Scientists have long desired to find a viable replacement for donated human organs and may have one in CRISPR modified pig organs.  Pig organs are roughly the same size and shape as human organs, however the presence of viruses in the pig genome have proven a significant barrier to use in clinical settings.  Now a company founded by George Church has used CRISPR to inactivate these viruses and generated 15 pigs that could be viable stocks for kidney, liver, lungs, or pancreas transplants.

Cellectis Granted CRISPR/Cas Patents for T-Cell Modification

Business Wire, 13 February 2018,

Cellectis has been granted patents for a T-cell genetic engineering method using transient expression of the CRISPR/Cas9 components.  A similar patent has already been issued by the European Patent Office.  This technology will be used to develop engineered CAR T-cells and the technology will be available for licensing.

Allele Specific CRISPR Editing

Sharon Begley, 02 February 2018, STAT,

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 (

Increasing Homology Directed Repair Efficiency by “Cold Shock”

Guo, Q., et. al. (2018) Scientific Reports. 8:2080.

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.