Gene Editing, A Revolution in Plant Breeding Prof. Dr. em. Klaus - PowerPoint PPT Presentation

Invited lecture, Prof. Dr. Behzad Ghareyazie, Founder of ABBRI Gene Editing, A Revolution in Plant Breeding Prof. Dr. em. Klaus Ammann, University of Bern, Switzerland Tehran, Iran, May 30, 2016 ABRII Agriculture Biotechnology Research Institute of Iran

The basic monograph, a draft literature review Ammann Klaus. (20160514). Modern Plant Breeding and Future Biosafety Regulation, 750 references with full text links for private use. ASK- FORCE Manuscript, pp. 323. http://www.ask-force.org/web/Genomic- Misconception/Ammann-Modern-Plant- Breeding-and-Future-Regulation- 20160514.pdf

Fig. 31 BACTERIA MAY NOT ELICIT MUCH Sympathy from us eukaryotes, but they, too, can get sick. That’s potentially a big probl em for the dairy industry, which often depends on bacteria such as Streptococcus thermophilus to make yogurts and cheeses. S. thermophilus breaks down the milk sugar lactose into tangy lactic acid. But certain viruses — bacteriophages, or simply phages — can debilitate the bacterium, wreaking havoc on the quality or quantity of the food it helps produce. In 2007, scientists from Danisco, a Copenhagen-based food ingredient company now owned by DuPont, found a way to boost the phage defenses of this workhouse microbe. They exposed the bacterium to a phage and showed that this essentially vaccinated it against that virus (Science, 23 March 2007, p. 1650). The trick has enabled DuPont to create heartier bacterial strains for food production. It also revealed something fundamental: Bacteria have a kind of adaptive immune system, which enables them to fight off repeated attacks by specific phages. Out of the text of (Pennisi, 2013) explaining the front figure.

Fig. 34 DNA surgeon. With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9 modifications have made possible the repression (lower left) or activation (lower right) of specific genes. From (Pennisi, 2013). Pennisi, E. (2013). The CRISPR Craze. Science, 341(6148), pp. 833-836. <Go to ISI>://WOS:000323370600011 AND http://www.ask- force.org/web/Genomics/Pennisi-CRISPR- Craze2013.pdf

Rapid Trait Development System ( RTDS) in Plants, from: http://www.cibus.com/technology.php

Rapid Trait Development System ( RTDS) in Plants, from: http://www.cibus.com/technology.php

Fig. 35 The Cas9 enzyme (blue) generates breaks in double-stranded DNA by using its two catalytic centers (blades) to cleave each strand f a DNA target site (gold) next to a PAM sequence (red) and matching the 20-nucleotide sequence (orange) of the single guide RNA (sgRNA). The sgRNA includes a dual- RNA sequence derived from CRISPR RNA (light green) and a separate transcript (tracrRNA, dark green) that binds and stabilizes the Cas9 protein. Cas9-sgRNA – mediated DNA cleavage produces a blunt double-stranded break that triggers repair enzymes to disrupt or replace DNA sequences at or near the cleavage site. Catalytically inactive forms of Cas9 can also be used for programmable regulation of transcription and visualization of genomic loci. From (Doudna & Charpentier, 2014) Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR- Cas9. Science, 346( 6213), pp. 1077-+. <Go to ISI>://WOS:000345763400031 AND http://www.askforce. org/web/Genomics/Doudna- Charpentier-New-Frontier-CRISP- Cas9-2014.pdf

CRISPR-Cas9 applications in plants and fungi also promise to change the pace and course of agricultural research. Future research directions to improve the technology will include engineering or identifying smaller Cas9 variants with distinct specificity that may be more amenable to delivery in human cells. Understanding the homology-directed repair mechanisms that follow Cas9-mediated DNA cleavage will enhance insertion of new or corrected sequences into genomes. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy.

Fig. 36 Diversity of CRISPR – Cas systems. The CRISPR- associated (Cas) proteins can be divided into distinct functional categories as shown. The three types of CRISPR – Cas systems are defined on the basis of a type- specific signature Cas protein (indicated by an asterisk) and are further subdivided into subtypes. The CRISPR ribonucleoprotein (crRNP) complexes of type I and type III systems contain multiple Cas subunits, whereas the type II system contains a single Cas9 protein. Boxes indicate components of the crRNP complexes for each system. The type III-B system is unique in that it targets RNA, rather than DNA, for degradation. From (van der Oost et al., 2014) Fig. 37 Architecture of crRNP complexes. a | Schematic representation of the subunit composition of different CRISPR ribonucleoprotein (crRNP) complexes from all three CRISPR – Cas types. The colours indicate homology with conserved Cas proteins or defined components of the complexes, as shown in the key. The numbers refer to protein names that are typically used for individual subunits of each subtype (for example, subunit 5 of the type I-A (Csa) complex refers to Csa5, Van-den-Oost et all managed to unravel the structural and mechanistic basis of CRISPR-Cas9- whereas subunit 2 of the type I-E (Cse) complex refers to Cse2, and so systems and give lots of very instructive figures. on). The CRISPR RNA (crRNA) is shown, including the spacer (green) and the flanking repeats (grey). Truncated Cas3 domains (Cas3ʹ van der Oost, J., Westra, E. R., Jackson, R. N., & Wiedenheft, B. (2014). Unravelling the structural and mechanistic basis of and Cas3ʹʹ) have been CRISPR-Cas systems. Nature Reviews Microbiology, 12( 7), pp. 479-492. <Go to ISI>://WOS:000338427600010 AND suggested to be part of the type I-A complex127, and fusions of http://www.ask-force.org/web/Genomics/VanderOost-Unravelling-structural-mechanistic-basis-CRISPR-Cas-systems2014.pdf Cas3 with

Fig. 37 Architecture of crRNP complexes. a | Schematic representation of the subunit composition of different CRISPR ribonucleoprotein (crRNP) complexes from all three CRISPR – Cas types. The colours indicate homology with conserved Cas proteins or defined components of the complexes, as shown in the key. The numbers refer to protein names that are typically used for individual subunits of each subtype (for example, subunit 5 of the type I-A (Csa) complex refers to Csa5, whereas subunit 2 of the type I-E (Cse) complex refers to Cse2, and so on). The CRISPR RNA (crRNA) is shown, including the spacer (green) and the flanking repeats (grey). Truncated Cas3 domains (Cas3ʹ and Cas3ʹʹ) have been suggested to be part of the type I-A complex127, and fusions of Cas3 with Cascade subunits (for example, with Cse1 (REF. 103)) have been found in some type I-E systems (shown as a dashed Cas3 homologue). Cas9 is depicted in complex with single-guide RNA (sgRNA), with an artificial linker (light grey) between the crRNA and the tracrRNA . Subunits with a RAMP (that is, an RNA-recognition motif (RRM)) fold are shown with a bold outline. The grey subunit in the type III-A Csm complex has been proposed to be a Cas7 homologue78. b | Structural comparison of crRNP complexes (colours as in part a): cryo- electron microscopy (cryo-EM) structures of Escherichia coli Cascade/I-E bound to a crRNA (two views after 90 ° rotation; Electron Microscopy Data Bank (EMDB) accession 5314; 8.8 Å)74, with additional double-stranded DNA (dsDNA) target (9 Å)89 and with additional Cas3 (20 Å)89. Cryo-EM structure of Streptococcus pyogenes Cas9 (of the type II-A system) bound to a single-guide RNA (sgRNA; not shown) and a 20 nucleotide target single-stranded DNA (ssDNA; not shown) (EMDB accession 5860; 21 Å), revealing a recognition lobe and a nuclease lobe, with a cleft in which the crRNA – DNA hybrid is located (see crystal structure; Supplementary information S2 (figure)). Cryo-EM structure of type III crRNP complexes: Sulfolobus solfataricus Csm complex (EMDB accession 2420; 30 Å)78, and Cmr complexes from Pyrococcus furiosus (EMDB accession 5740; 12 Å)79 and Thermus thermophilus69. From (van der Oost et al., 2014 “Abstract | Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR– Cas (clustered regularly interspaced short palindromic repeats – CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR – Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea. ” From (van der Oost et al., 2014)