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A NEW ERA OF GENETIC MANIPULATION

Posted in Academic Publishing, Bacterial Resistance, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Genetics & Pharmaceutical, Genome Biology, tagged biomarkers, Cas9, CRISPR, CRISPR/Cas9, Demet Sag, Dr. Sag, functional genomics, Functional TransGenomics, gene editing, genome structure and function, germline, Immunology, immunomodulation, innate immune defense, manipulation of gene, Microbiology, scientific, stem cels, translational genomics on April 14, 2015| Leave a Comment »

Demet Sag, PhD, CRA, GCP

Gene engineering and editing specifically are becoming more attractive. There are many applications derived from microbial origins to correct genomes in many organisms including human to find solutions in health.

There are four customizable DNA specific binding protein applications to edit the gene expression in translational genomics. The targeted DNA double-strand breaks (DSBs) could greatly stimulate genome editing through HR-mediated recombination events. We can mainly name these site-specific DNA DSBs:

meganucleases derived from microbial mobile genetic elements (Smith et al., 2006),
zinc finger (ZF) nucleases based on eukaryotic transcription factors (Urnov et al., 2005;Miller et al., 2007),
transcription activator-like effectors (TALEs) from Xanthomonasbacteria (Christian et al., 2010; Miller et al., 2011; Boch et al., 2009; Moscou and Bogdanove, 2009), and
most recently the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (Cong et al., 2013;Mali et al., 2013a).

There is a new ground breaking study published in Science by Valentino Gantz and Ethan Bier of the University of California, San Diego, described an approach called mutagenic chain reaction (MCR).

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

Structural and Metagenomic Diversity of Cas9 Orthologs

(A) Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA.

(B) Canonical CRISPR locus organization from type II CRISPR systems, which can be classified into IIA-IIC based on their cas gene clusters. Whereas type IIC CRISPR loci contain the minimal set of cas9, cas1, andcas2, IIA and IIB retain their signature csn2 and cas4 genes, respectively.

(C) Histogram displaying length distribution of known Cas9 orthologs as described in UniProt, HAMAP protein family profile MF_01480.

(D) Phylogenetic tree displaying the microbial origin of Cas9 nucleases from the type II CRISPR immune system. Taxonomic information was derived from greengenes 16S rRNA gene sequence alignment, and the tree was visualized using the Interactive Tree of Life tool (iTol).

(E) Four Cas9 orthologs from families IIA, IIB, and IIC were aligned by ClustalW (BLOSUM). Domain alignment is based on the Streptococcus pyogenes Cas9, whereas residues highlighted in red indicate highly conserved catalytic residues within the RuvC I and HNH nuclease domains.

(Cell. Author manuscript; available in PMC 2015 Feb 27.Published in final edited form as:

Cell. 2014 Jun 5; 157(6): 1262–1278.doi: 10.1016/j.cell.2014.05.010)

The uniqueness of this study comes from:

There is a big difference between the new type of mutation and traditional mutation is expressivity of the character since previously mutations were passive and non-transferable at 100% rate. However, in classical Mendelian Genetics, only one fourth f the recessive traits can be presented in new generation. Yet, in this case this can be achieve about 97% plus transferred to new generation.

MCR alterations is active that is they convert matching sequences at the same target site so mutated sites took over the wild type character without degenerating by wild type alleles segregating independently during the breeding process

Therefore, the altered sequences routinely replace the wild type (original) sequences at that site. The data demonstrated that among 92 flies, only one female became wild type but remaining 41 females had yellow eyes yet all 50 males showed wild type eye coloring at the second generation.

The genetic engineering of the genome occurred in a single generation with high efficiency.

Their technique developed by Gantz and Bier had three basic parts:

Both somatic and germline cells expressed a Cas9 gene,

A guide RNA (gRNA) targeted to a genomic sequence of interest,

The Cas9/gRNA cassettes have the flanking homolog arms that matches the two genomic sequences immediately adjacent to either side of the target cut site

There are many applications in translational genomics that requires multiple steps to make it perfect for complicated organisms, such as plants, mosquitoes and human diseases.

Short Walk from Past to the Future of CRISPR/Cas9

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner.

The latest ground-breaking technology for genome editing is based on RNA-guided engineered nucleases, which already hold great promise due to their:

simplicity,
efficiency and
versality

Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987),

their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005).

Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007).

The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

The most widely used system is the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes (Jinek et al., 2012).

Then, five independent groups demonstrated that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013, Cong et al., 2013, Hwang et al., 2013, Jinek et al., 2013 and Mali et al., 2013).

Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified colonial cell lines can be derived within 2-3 weeks

Genome editing with site-specific nucleases.

Double-strand breaks induced by a nuclease at a specific site can be repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR). In most cases, NHEJ causes random insertions or deletions (indels), which can result in frameshift mutations if they occur in the coding region of a gene, effectively creating a gene knockout.

Alternatively, when the DSB generates overhangs, NHEJ can mediate the targeted introduction of a double-stranded DNA template with compatible overhangs

Even though the generation of breaks in both DNA strands induces recombination at specific genomic loci, NHEJ is by far the most common DSB repair mechanism in most organisms, including higher plants, and the frequency of targeted integration by HR remains much lower than random integration.

Unlike its predecessors, the CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene

Unlike ZFNs and TALENs, the CRISPR/Cas9 system can cleave methylated DNA in human cells (Hsu et al., 2013), allowing genomic modifications that are beyond the reach of the other nucleases (Ding et al., 2013).

The main practical advantage of CRISPR/Cas9 compared to ZFNs and TALENs is the ease of multiplexing. The simultaneous introduction of DSBs at multiple sites can be used to edit several genes at the same time (Li et al., 2013; Mao et al., 2013) and can be particularly useful to knock out redundant genes or parallel pathways.

Finally, the open access policy of the CRISPR research community has promoted the widespread uptake and use of this technology in contrast, for example, to the proprietary nature of the ZFN platform.

The community provides access to plasmids (e.g., via the non-profit repository Addgene), web tools for selecting gRNA sequences and predicting specificity:

http://cbi.hzau.edu.cn/ cgi-bin/CRISPR; http://www.genome.arizona.edu/crispr/; and http:// http://www.rgenome.net/cas-offinder,
http://www.e-crisp.org/E-CRISP/index. html
hosts active discussion groups (e.g.: https://groups.google. com/forum/#!forum/crispr).

Downside:

One area that will likely need to be addressed when moving to more complex genomes, for instance, is off-target CRISPR/Cas9 activity since fruit fly has only four chromosomes and less likely to have off-target effects. However, this study provided proof of principle.

Yet, this critics is not new since one of the few criticisms of the CRISPR/Cas9 technology is the relatively high frequency of off-target mutations reported in some of the earlier studies (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jiang et al., 2013a; Mali et al., 2013; Pattanayak et al., 2013).

Several strategies have been developed to reduce off-target genome editing, the most important of which is the considered design of the gRNA.

fusions of catalytically inactive Cas9 and FokI nuclease have been generated, and these show comparable efficiency to the nickases but substantially higher (N140-fold) specificity than the wild-type enzyme (Guilinger et al., 2014; Tsai et al., 2014)

Altering the length of the gRNA can also minimize non-target modifications. Guide RNAs with two additional guanidine residues at the 5′ end were able to avoid off-target sites more efficiently than normal gRNAs but were also slightly less active at on-target sites (Cho et al., 2014)

What more:

The CRISPR/Cas9 system can be used for several purposes in addition to genome editing:

The ectopic regulation of gene expression, which can provide useful information about gene functions and can also be used to engineer novel genetic regulatory circuits for synthetic biology applications.

The external control of gene expression typically relies on the use of inducible or repressible promoters, requiring the introduction of a new promoter and a particular treatment (physical or chemical) for promoter activation or repression.

Disabled nucleases can be used to regulate gene expression because they can still bind to their target DNA sequence. This is the case with the catalytically inactive version of Cas9 which is known as dead Cas9 (dCas9).

Preparing the host for an immunotherapy is possible if it is combined with TLR mechanism:

On the other hand, the host mechanism needs to be review carefully for the design of an effective outcome.

The mechanism of microbial response and infectious tolerance are complex.

During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells.

Uniqueness of TLR comes from four major characteristics of each individual TLR :

ligand specificity,
signal transduction pathways,
expression profiles and
cellular localization.

Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels Specific TLR stimulat ion links innate and acquired responses through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.

Some examples of ligand – TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2, double stranded (ds) RNAs through TLR3, lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5, single stranded RNAs through TLR7/8, synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9.

The specificity is established by correct pairing of a TLR with its proinflammatory cytokine(s), so that these permutations influence creation and maintenance of cell differentiat ion.

Immunotherapy: The immune cells can be used as a sensor to scavenger the circulating malformed cells in vivo diagnostics or attack and remember them, for instance, relapse of cancer, re-infection with a same or similar agent (bacteria or virus) etc.

Not only using unique microbial and other model organism properties but also using the human host defense mechanism during innate immune responses may bring a new combat to create a new method of precision medicine. This can be a new type of immunotherapy, immune cell mediated gene therapy or vaccine even a step for an in vivo diagnostics.

Molecular Genetics took a long road from discovery of restriction enzymes, developing PCR assays, cloning were the beginning. Now, having technology to sequence and compare the sequences between organisms also help to design more sophisticated methods.

Generating mutant lines in Drosophila with the classical genetics methods relies on P elements, a type of transposon and balancers after crossing selected flies with specific markers. This fly pushing is a very tedious work but powerful to identify primary pathways, mechanisms and gene interactions in system and translational genomics.

Thus, Microbial Immunomodulation is an important factor not only using the microorganisms or their mechanisms but also modulating the immune cells based on the host interaction may generate new types of diagnostics and targeted therapy tools.

Microbial immunomodulation. Microbes from the environment, and from the various microbiota, modulate the immune system. Some of this is due to direct effects of defined microbial products on elements of the immune system. But modulation of the immune system also secondarily alters the host–microbiota relationship and leads to changes in the composition of the microbiota, and so to further changes in immunoregulation (shown as indirect pathways). At the end of the day balance is the key for survival.

Graham A. W. Rook,^*,1 Christopher A. Lowry,² and Charles L. Raison³Microbial ‘Old Friends’, immunoregulation and stress resilience Evol Med Public Health. 2013; 2013(1): 46–64. Published online 2013 Apr 9. doi: 10.1093/emph/eot004 PMCID: PMC3868387

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881665/bin/nihms199923f2.jpg

CRISPR-Cas9 mediated NHEJ in transient transfection experiments.

Table 1.

Species	Transformation method	Cas9 codon optimization	Promoters (Cas9, gRNA)	Target	Mutation frequency	Detection method	Off-target (no. of sites analyzed)	Detection method	Multiplex (deletion)	Reference
Arabidopsis thaliana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3<comma> FLS2	1.1–5.6%	PCR + sequencing				Li et al. (2013)
A. thaliana	Leaf agroinfiltration	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	2.70%	PCR + sequencing			Yes (48 bp)	Li et al. (2013)
A. thaliana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	RACK1b<comma> RACK1c	2.5–2.7%	PCR + sequencing	No (1 site)	PCR + sequencing		Li et al. (2013)
A. thaliana	Leaf agroinfiltration	C. reinhardtii	CaMV35S, AtU6	Co-transfected GFP	n.a.	Pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
Nicotiana benthamiana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	37.7–38.5%	PCR + sequencing				Li et al. (2013)
N. benthamiana	Leaf agroinfiltration	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	4.80%	PCR + sequencing				Li et al. (2013)
N. benthamiana	Leaf agroinfiltration	Human	CaMV35S, AtU6	PDS	1.8–2.4%	PCR + RE	No (18 sites)	PCR + RE		Nekrasov et al. (2013)
N. benthamiana	Leaf agroinfiltration	C. reinhardtii	CaMV35S, AtU6	Co-transfected GFP	n.a.	pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
N. benthamiana	Leaf agroinfiltration	Human	CaMV35S, CaMV35S	PDS	12.7–13.8%					Upadhyay et al. (2013)
Nicotiana tabacum	PEG-protoplast transfection	Tobacco	2xCaMV35S, AtU6	PDS<comma> PDR6	16.27–20.3%	PCR + RE			Yes (1.8 kb)	Gao et al. (2014)
Oryza sativa	PEG-protoplast transfection	Rice	2xCaMV35S, OsU3	PDS<comma> BADH2<comma> MPK2<comma> Os02g23823	14.5–38.0%	PCR + RE	Noa (3 sites)	PCR + RE		Shan et al. (2013)
O. sativa	PEG-protoplast transfection	Human	CaMV35S, OsU3 or OsU6	MPK5	3–8%	RE + qPCR and T7E1 assay	No (2 sites) Yes (1 site with a mismatch at position 12)	RE + PCR		Xie and Yang (2013)
O. sativa	PEG-protoplast transfection	Rice	CaMV35S, OsU6	SWEET14	n.a.	pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
O. sativa	PEG-protoplast transfection	Rice	ZmUbi, OsU6	KO1 KOL5; CPS4 CYP99A2; CYP76M5 CYP76M6	n.a.	PCR + sequencing			Yes (115<comma> 170<comma> 245 kb)	Zhou et al. (2014)
Triticum aestivum	PEG-protoplast transfection	Rice	2xCaMV35S, TaU6	MLO	28.50%	PCR + RE				Shan et al. (2013)
T. aestivum	PEG-protoplast transfection	Plant	ZmUbi, TaU6	MLO-A1	36%	T7E1				Wang et al. 2014a and Wang et al. 2014b
T. aestivum	Agrotransfection of cells from immature embryos	Human	CaMV35S, CaMV35S	PDS<comma> INOX	18–22%	PCR + sequencing				Upadhyay et al. (2013)
T. aestivum	Agrotransfection of cells from immature embryos	Human	CaMV35S, CaMV35S	INOX		PCR + sequencing	No*	PCR + RE	Yes (53 bp)	Upadhyay et al. (2013)
Zea mays	PEG-protoplast transfection	Rice	2xCaMV35S, ZmU3	IPK	16.4–19.1%	PCR + RE				Liang et al. (2014)
Citrus sinensis	Leaf agroinfiltration	Human	CaMv35S, CaMV35S	PDS	3.2–3.9%	PCR + RE	No (8 sites)	PCR + RE		Jia et al. (2014)

References:

A brief overview of CRISPR-mediated immunity and explain how the emerging new properties of this defense system are being repurposed for genome engineering in bacteria, yeast, human cells, insects, fish, worms, plants, frogs, pigs, and rodents.

Also look at F1000Prime Rep. 2014; 6: 3. For the list of microorganisms use in CRISPR applications.

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Previously Published at Leaders in Pharmaceutical Intelligence:

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

larryhbern

2015/03/27
Published

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com

Posts Tagged ‘innate immune defense’

A NEW ERA OF GENETIC MANIPULATION

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