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.

Tags: Caribou, cas9, CRISPR, Intellia, iPSC, Novartis, SBIR, TALEN, ZFN

Genome Modification—a Practical Approach

The ability to modify genomes has always been fervidly sought after by molecular, developmental biologists and geneticists as it would provide them with the means for finding out what a particular piece of the genome may do in the biological process they are studying. The discovery of naturally existing P-element helped a generation of Drosophila geneticists and made the fruit fly a prime model system for gene function studies in the 80’s and 90’s. But P-elements inserted at uncontrolled sites, making it essentially a gene transfer vehicle without much control. The introduction of prokaryotic recombination systems, e.g. LoxP and Cre, provided researchers with tools to obtain more control of the inserted genes in a host chromosome during a biological process such as development. Transposons like Sleeping Beauty, Piggybac, or Tol2 made similar experiments possible in mammalian cells.

Still, the randomness of transposon-type elements’ insertion, much like retrovirus or lentivirus, could cause trouble if they land in an undesirable spot. Methods of inserting transgenes only in well-known, harmless, and transcriptionally active regions, so called “safe harbors”, were subjects of interest of researchers and NIH grant topics in the past couple of years under “directed genome editing”. Gene knock-out or knock-in can be achieved through vector-mediated homologous recombination such as the rAAV genome engineering system and the “TARGATT” system, which are commercially available as kits or services.

However, instead of inserting an exogenous gene, it is often highly desirable to modify an endogenous genome sequence, which requires the modification apparatus to first recognize the target sequence. ZFN and TALEN both recognize DNA targets through specific nucleotide binding protein domains, with TALEN having more flexibility if assembled in a “Lego”-like format because each domain can specifically recognize a “C”, “G”, “T”, or “A” base. The description of using CRISPR/cas system in a recent burst of publications opened up new ways of binding to specific DNA sequences and nicking or severing the dsDNA. This system does not require engineeredDNA binding domain assembly; instead, it uses a guide RNA to find the target DNA sequence to direct endonuclease, in a sense quite like RNAi. However, the enthusiasm about CRISPR/cas was somewhat dampened by a report last month in Nature Biotechnology that reported off-target effects of CRISPR/cas was much higher than ZFN and TALEN. Particularly, if mismatches are located in the 5’ portion of the guide RNA targeting sequence, they can be well tolerated up to 3 or 4, even 5 mismatches. Unfortunately this is also similar to the tolerance of the RNAi matching region outside the core 12-base region. The difference is: for RNAi, the off-target damage is temporary and ignorable if the extent is insignificant compared to the effects on the intended target while for CRISPR/cas, an off-target cut on the chromosome is permanent.

On the positive side, in an even more recent publication in Nature Methods, mutant strains of C. elegans were obtained using the CRISPR/cas system and no evidence was found for off-target changes, at least not in an overwhelming fashion. Much value of the estimates of off-target effects relates to the methods used for analysis. Currently, most of the studies looked at potential off-target sides by searching for partial matches. In the future, whole genome sequencing will be increasingly required for submitting such publications.

On a practical note, if you intend to take a dive and try to use any one of these methods, your number one problem will be that none of the methods will result in 100% modification even if you can ignore the off-target problems for now. Therefore, many of our customers ask about a screening strategy. One could use traditional drug selection and fluorescent protein (FP)-based sorting, but these can only help you find cells that are successfully transfected with the ZFN, TALEN, or CRISPR/cas expressing DNA molecules, not necessarily having the genome modification result. We have formulated the idea of inserting the target site into an FP-bearing plasmid as a surrogate target cutting indicator, and use another FP to track transfection of the TALEN plasmid. Nonetheless, in the end, PCR-amplifying the target region of the chromosome and doing either an enzymatic mismatch detection assay (e.g. T7 endonuclease) or sequencing is the only way to know for sure whether genome editing has occurred.

Tags: cas, cre/loxP, CRISPR, CRISPR 101, genome engineering, genome sequencing, P-element, Piggybac, rAAV, safe harbor, Sleeping Beauty, TALEN, TARGATT, Tol2, ZFN

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.

Tags: cas9, choromsome modification, CRISPR/cas, F. novicida, genome engineering, nuclease, RNA interference, RNAi, S. pyogenes, TALE, TALEN, ZFN

Conducting Massively Parallel Sequencing

One of the major breakthroughs in modern biology is the development of massively parallel sequencing, also called next generation sequencing (NGS), which enabled the complete delineation of the human genome more than a decade ago. Since then many more species’ genomes have been sequenced, and the cost per genome has dropped from billions to mere thousands of dollars. New discoveries are being made as a result of the capability many research teams now possess to not only sequence chromosomal DNA, but also to identify which regions a protein of interest specifically binds (Chip-seq), analyze a whole transcriptome of a cell population under investigation (RNA-seq), or find out which RNA regions an RNA binding protein resides (CLIC-seq).

While it is inevitable that many PIs will seriously consider the inclusion of deep sequencing in their next grant proposal, it is not necessarily easy to take the first step and get their feet wet, so to speak. Knowing what format (e.g. 454 for longer reads, HighSeq for higher accuracy, or Ion Torren for bench top convenience) to use and how much to pay requires a vast amount of knowledge and experience. Even when you are done with sample prep, amplification and sequencing, to handle such massive amount of data is not trivial—transporting data alone can be a headache. A database server for storage and analysis requires another layer of expertise. There is no easy solution but to get started somehow. However, be prepared to deal with these issues.

Whether the cost on a type of next generation service is justifiable depends on whether it is required for your purposes. For example, when analyzing a person’s propensity of developing a disease by using known, disease-relevant genetic information, often times exome sequencing is sufficient. This costs anywhere between $1,000 to $3,000 with 100X coverage, significantly less than sequencing a complete genome which typically costs ~$5,000 at ~20x coverage.

High coverage sequencing of maternal blood DNA has been developed into clinically approved prenatal diagnosis of trisomy in Down’s syndrome and other chromosomal abnormalities. Transcriptome analysis helped the understanding of how reprogramming works when iPSCs are. Looking forward, with more routine use of deep sequencing we can predict with much more certainty the “off-target” effects of RNAi or cellular toxicity of chromosomal modifications enabled by ZFN, TALEN, or CRISPR. As a matter of fact, we believe that transcriptome sequencing should be required after each RNAi event to prove a specific linkage between knockdown and functions; similarly, whole genome sequencing results need to be provided after making a site directed chromosomal change in the future for high level publications.

*This blog partially resulted from discussions between Jiwu Wang and his colleagues, who are NGS experts at UCSD’s Cellular and Molecular Medicine, Moore Cancer Center, and BGI Americas.

Tags: CRISPR, deep sequencing, exome sequencing, footprint free, genomic modification, iPSCs, Massively Parallel Sequencing, NSG, TALEN, transcriptome, whole genome sequencing, ZFN

Legoland TALES of Binding DNA by Design

Imagine that you could have a protein that binds to a sequence in a chromosome where you want to activate transcription, nick or break DNA to target insertion or recombination, or create a DNA lesion for screening DNA repair pathway factors…imagine that you could build such proteins very much like putting together legos. Yes, it is possible based on findings about transcription activator-like effector (TALE) proteins. These are plant pathogen transcription factors naturally used to facilitate invasion of host species which have been rekindled to direct DNA binding in other species (1, 2).

Previously, zinc-finger nucleases (ZFNs) have been the focus of genomic modification tool development, but with only limited success. It is not easy to design or select a ZFN using available technologies. In comparison, TALEs have a modular 34 amino acid domain as a basic unit that recognizes a DNA base, with specificity mostly determined by residues 12 and 13. In other words, by using as few as 4 modules with dedicated diamino acids 12 and 13, one can create a protein that binds any DNA sequence.

However, it is not necessarily an easy construct to make because the highly repetitive sequence of TALEs causes plasmid instability during cloning. A team at Harvard recently published a method of minimizing repetitiveness and allowing step-wise ligation; (3). Other aspects of using TALEs involve the designing of an effector domain, e.g. DNAase or transcription activation domain, and packaging the “warhead” in a delivery vehicle such as a lentivirus. The unfolding of the TALEs is just starting, the future seems exciting.

1. J. Boch et al. Science 326, 1509 (2009); published online 29 October 2009
2. M. J. Moscou, and A. J. Bogdanove, Science 326, 1501 (2009)
3. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

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Tags: DNA binding, DNA repari, homologues recombination, modular, nuclease, TALE, TALEs, transcription activator, ZFN, ZFNs