cas9

Allele’s SBIR Grant to Develop All-RNA CRISPR

Precise engineering of the genomes of mammalian cells enabled biological and medical applications researchers had dreamed of for decades. Recent developments in the stem cell field have created even more excitement for genetically modifying genomes because it enables delivering more beneficial stem cell-derived therapeutic cells to patients [1]. For instance, by correcting a gene mutation known to be critical to Parkinson’s disease, LRRK2 G2019S, in patient-specific iPSCs (induced pluripotent stem cells), it appeared possible to rescue neurodegenerative phenotypes [2].

Significant amount of fund and energy had been invested in technologies such as ZFN and TALEN, however, judging from the explosion of publications and business activities in just about 2 years since the illustration of its mechanism (just today, Jan 8th, 2015, Novartis announced CRISPR collaborations with Intellia, Caribou, applying it in CAR T cell and HSCs), the CRISPR/cas system is the rising star. This system uses a guide RNA to direct the traffic of a single nuclease towards different targets on a chromosome to alter DNA sequence through cutting. The nuclease, cas9, can be mutated from a double-stranded DNA endonuclease to a single-strand cutter or a non-cutting block, or further fused to various functional domains such as a transcription activation domain. This system can also be used to edit RNA molecules.

A weak spot on the sharp blade of CRISPR is, like any methods for creating loss-of-function effects (RNAi if you remember), the potential of off-target effects. While they can never be completely avoided, with the ever growing popularity of deep sequencing, at least we can know all unintended changes on the edited genome. Almost a perfect storm! As an interesting side story, when we at Allele Biotech first saw the paper in Science describing the CIRPSR/cas system [3], we immediately wrote an SBIR grant application for applying the bacterial system to mammalian cells. The first round of review in December 2012 concluded that it would not work due to eukaryotes’ compact chromatin structures. Of course, the flurry of publication in early 2013, while our application was being resubmitted, proved otherwise. The good news is, Allele Biotech still received an SBIR grant from NIGMS in 2014. Unlike most of the genome editing platforms known in the literature, our goal was to build an all-RNA CRISPR/cas system, thereby with higher potency, less off-target effects, and, as a footprint-free platform, more suitable for therapeutic applications. This system will be combined with our strengths in iPSC and stem cell differentiation, fluorescent protein markers, and deep sequencing based bioinformatics to improve cell therapy and cell based assays.

1 Urnov, F.D., et al., Genome editing with engineered zinc finger nucleases. Nat Rev Genet, 2010. 11(9): p. 636-46.
2 Reinhardt, P., et al., Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression. Cell Stem Cell, 2013. 12(3): p. 354-67.
3 Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.

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Thursday, January 8th, 2015 Allele Mail Bag, iPSCs and other stem cells, Next Generation Sequencing (NextGen Seq), NIH Budget and You, SBIR and Business issues 1 Comment

The Power of Cas

Precise engineering of the genomes of higher eukaryotes can enable a variety of biological and medical applications. Targeted gene disruption, editing, and insertion can translate into the much desired freedom to generate cells or organisms bearing a desired genetic change. Recent developments in the stem cell field have created even more excitement for genetically modifying genomes because it enables delivering more beneficial stem cell-derived therapeutic cells to patients. For instance, by correcting a gene mutation known to be critical to Parkinson’s disease, LRRK2 G2019S, in patient-specific iPSCs (induced pluripotent stem cells), researchers were able to rescue neurodegenerative phenotypes [1].

Cumbersome reagent development and high costs have been major barriers to targeted genome modification using the current technologies, which include the zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Unlike the ZFN and TALEN systems, CRISPR/cas does not require assembly of DNA pieces that encode the functional proteins every time a new sequence is to be targeted. Instead, it uses a guide RNA to direct the traffic of a nuclease complex. Five recent publications of modifying eukaryotic chromosomes showed the importance of the CRISPR/cas system [2-6], they also hinted at the ease of adapting this system in eukaryotes given that the functions of cas and the small guide RNA were described in bacteria merely few months ago [7].

The concern that the bacterial CRISPR/cas system would not access the chromatin structures of eukaryotic genome was muted as a result of recent publications; it also seems that the cas9 protein is as powerful an enzyme as one could have hoped in an endonuclease. As a matter of fact, cas9 from S. pyogenes contains 2 different single-stranded DNAse domains independent of each other, and can be mutated to change from a double-stranded DNA endonuclease to a single-strand cutter, or a non-cutting block. That’s not all, a more recent Nature publication further showed that cas9 (from another species, F. novicida), can bind to yet another small RNA and, instead of cutting chromosomal DNA, it degrades RNA, apparently through a direct cas9/RNA binding mechanism [8]. It may be chromosomal modification and RNAi rolled in one (cas9 from different genera are quite different though). One has to admire the powerful cas!

1. Reinhardt, P., et al., Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression. Cell Stem Cell, 2013. 12(3): p. 354-67.
2. Qi, L.S., et al., Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 2013. 152(5): p. 1173-83.
3. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.
4. Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.
5. Cho, S.W., S. Kim, J.M. Kim, and J.S. Kim, Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013. 31(3): p. 230-2.
6. Hwang, W.Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(3): p. 227-9.
7. Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.
8. Sampson, T.R., et al., A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 2013.

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Thursday, May 2nd, 2013 Next Generation Sequencing (NextGen Seq), Synthetic biology No Comments