1 Редактирование геномов растений

2 ТЕХНОЛОГИИ, ИЗМЕНИВШИЕ ГЕНЕТИКУКлеточная система репарации геномной ДНК, сайт-специфичные нуклеазы (ZFN, TALEN) и РНК-направляемая нуклеаза Cas. Современная технология геномного редактирования основана на возможности инициировать разрыв двуцепочечный молекулы ДНК (DSB) в определённых/специфичных местах и затем соединять образованные концы (восстанавливать разрыв ДНК) с помощью собственной клеточной системы репарации. При этом может происходить соединение негомологичных концов (NHEJ, что приводит к образованию делеций или вставок нуклеотидов, и как результат – инактивация гена), а если присутствует ДНК с гомологичнными концевым последовательностями, то будет происходить гомологичная рекомбинация (HDR, homology directed repaire) со встраиванием последовательности, находящейся между гомологичными фрагментами. Эффективная инициация сайт-специфичных DSB стала возможна после создания искусственных нуклеаз, состоящих из ДНК-связывающего домена и нуклеазы FokI. Цинковые пальцы узнают триплет, а дипептиды – отдельный основания

3 Сайт-специфичные нуклеазыА, В – специфичность определяется белком. Трудоёмкий, недешёвый дизайн. С – специфичность определяется коротким фрагментом РНК, легко менять.

4 Цинковые пальцы

5 Zink Fingers Figure 1. Schematic illustration of the ZFN structure and the principle of ZFN-mediated genomic modifications. The target site of the ZFN is recognized by the ‘‘left’’ and ‘‘right’’ half monomer that each consist of a tandem array of engineered ZFPs, and each engineered ZFP can recognize a nucleotide triplet (shown in different colors). The ZFN monomer is comprised of an N-terminal domain containing a NLS (red), a recognition domain that usually comprises tandem ZFPs (in different colors) and a C-terminal function domain that comprises the Fok I endonuclease. Recognition of the target sequence by the left and right ZFPs results in dimerization of the Fok I endonuclease, which is critical for the activity of the ZFNs. DNA cleavage takes place between the two ZFP recognition sites that contain a spacer sequence that is usually 6 bp long. Induced DSB of the target DNA are repaired either by NHEJ or HDR, resulting in gene mutation around the cleavage sites. NLS, nuclear localization signal; ZFP, zinc finger proteins; DSB, double-strand breaks; NHEJ, non-homologous end joining; HDR, homology-directed repair. Mutation*, the red color box region contains nucleotide deletion, insertion or substitution. Figure modified from Gaj et al. (2013), Figure 1 and Moore et al. (2012), Figure 1. 100

6 TALEN Figure 2. The structure of TALEN and the principle of TALEN-mediated genomic modifications. The target site of TALEN is recognized by the ‘‘left’’ and ‘‘right’’ half monomer that each consist of a tandem repeat of TALE repeats. Each TALE repeat comprises a 34 amino acid (aa) unit that differs at two hypervariable aa located at the 12th and 13th position, known as RVD, which determine the recognition specificity of each repeat. The TALEN monomer consists of an N-terminal domain containing a nuclear localization signal (NLS, red), a recognition domain typically composed of tandem TALE repeats (in different colors), and a C-terminal function domain that comprises the Fok I endonuclease. Simultaneous bindings of the left and right TALE enable dimerization of the Fok I cleavage domain, resulting in DSBs of the target DNA. Induced DSBs of the target DNA are repaired either by NHEJ or HDR resulting in gene mutations that include nucleotide insertion, deletion, or substitution around the cleavage site. TALE, transcription activator-like effector; NLS, nuclear localization signal; RVD, repeat-variable di-residues; DSB, double-strand breaks; NHEJ,non-homologous end joining; HDR, homology-directed repair. Mutation*, red color box regions contain nucleotide deletion, insertion or substitution. Figure modified from Gaj et al. (2013), Figure 1 and Moore et al. (2012), Figure 1. 100

7 CRISPR/Cas9- Figure 3. Schematic illustration of the CRISPR/Cas9 system structure and principle of CRISPR/Cas9-mediated genomic modifications. The synthetic guide RNA (sgRNA) contains a region (usually 20 bp in length) complementary to the target site on the genomic loci and stem loops that mediate the binding of the Cas9 protein. The protospacer adjacent motif (PAM, NGG) required for cleavage is indicated in red, the Cas9 protein is shown by the brown circle, and the cleavage sites located 3 bp from the PAM motif are indicated by scissors. Induced DSBs of the target DNA are repaired either by NHEJ or HDR resulting in gene mutations that include nucleotide insertion, deletion or substitution around the cleavage sites. sgRNA, synthetic guide RNA; DSB, double-strand breaks; NHEJ, non-homologous end joining; HDR, homology-directed repair. Mutation*, red color box region contains nucleotide deletion, insertion or substitution. Figure modified from Xie and Yang (2013), Figure 1. 76

8 Хронология научных исследований в области биологии CRISPR-CAS и геномной инженерииCRISPR biology Genome editing The transition of the CRISPR/Cas system from biological phenomenon to genome engineering tool came about when it was shown that the target DNA sequence could be reprogrammed simply by changing 20 nucleotides in the crRNA and that the targeting specificity of the crRNA could be combinedwith the structural properties of the tracrRNA in a chimeric single guide RNA (gRNA), thus reducing the system from three to two components (Jinek et al., 2012; Fig. 2b). Shortly thereafter, 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; Mali et al., 2013). Importantly, it was also shown that multiple gRNAs with different sequences could be used to achieve high-efficiency multiplex genome engineering at different loci simultaneously (Cong et al., 2013; Mali et al., 2013). These milestones confirmed that the CRISPR/Cas9 system was a simple, inexpensive and versatile tool for genome editing, resulting in a groundswell of research based on the technique which has become known as the ‘CRISPR craze’ (Pennisi, 2013). In August 2013, five reports were published discussing the first application of CRISPR/Cas9-based genome editing in plants ( 1985–1991 Zinc-finger proteins 1996–2003 Zinc-finger nucleases for genome engineering 2003 onward Expanded use of ZFNs for genome engineering 1979 Gene replacement in yeast 1985–1986 Human genome editing by HDR 1989–1994 Genome break repair by NHEJ, HDR 2009–2010 TAL effectors; TALE nucleases 2010 onward Increasing use of TALENs for genome engineering

9 Хронология научных исследований в области биологии CRISPR-CAS и геномной инженерии

10 CRISPR/Cas9- inspiration from the Mother Nature (система CRISPR/Cas – природный механизм адаптивного иммунитета бактерий и архей) clustered regularly interspaced short palindromic repeats - короткие палиндромные повторы, регулярно расположенные группами Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated

11 Просто, универсально, дёшево

12 РЕДАКТИРОВАНИЕ ГЕНОМОВ РАСТЕНИЙCo-transformation Agro-infiltration

13 CRISPR-Cas9 mediated NHEJ in transient transfection experiments

14 CRISPR-Cas9 mediated NHEJ in stable transformants

15 Homologous recombination using the CRISPR system

16 http://www. addgene. org/search/advanced/

17 The studies also confirmed that single chimeric gRNAs are more efficient than separate crRNA and tracrRNA components in plants, just as they are in other eukaryotes (Miao et al., 2013; Zhou et al., 2014). four independent groups have shown that the CRISPR/Cas9 system can introduce biallelic or homozygous mutations directly in the first generation of rice and tomato transformants, highlighting the exceptionally high efficiency of the system in these species (Brooks et al., 2014; Shan et al., 2013; Zhang et al., 2014; Zhou et al., 2014). It was also shown in Arabidopsis, rice and tomato that the genetic changes induced by Cas9/gRNA were present in the germ line and segregated normally in subsequent generations without further modifications

18 CRISPR-PLANT A Portal of CRISPR-Cas9 Mediated Genome Editing Available Datasets Arabidopsis thaliana Brachypodium distachyon Glycine max Medicago truncatula Oryza sativa Sorghum bicolor Solanum lycopersicum ВЫБОР gRNA (РНК-ГИД)

19 Improving cold storage and processing traits in potato through targeted gene knockoutTranscription activator-like effector nucleases (TALENs) has been used to knockout VInv within the commercial potato variety, Ranger Russet. 18 plants containing mutations in at least one VInv allele were isolated, and five of these plants had mutations in all VInv alleles. T Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome.

20 Targeting the Solanum tuberosum cv Ranger Russet VInv gene with transcription activator-like effector nucleases (TALENs) (a) During cold storage, potato tubers accumulate acrylamide through a nonenzymatic Maillard reaction, which uses reducing sugars (primarily glucose and fructose) and free amino acids (asparagine [Asn]). Reducing sugars accumulate through hydrolysis of sucrose by vacuolar acid invertase (VInv). In addition to accumulating acrylamide, cold-stored potatoes also produce brown- to black-pigmented products. (b) Schematic of the VInv gene. TALEN target sites are indicated with black, grey and white triangles (VInv_T1, VInv_T2, VInv_T3, respectively). (c) TALEN target sites within exon 1. Single nucleotide polymorphisms are indicated by lowercase bold letters. Underlined letters indicate TALEN-binding sites. A1, allele 1; A2(1), copy 1 of allele 2; A2(2) copy 2 of allele 2; A(3), allele 3.

21 Оценка активности TALEN в протопластах Solanum tuberosum cv Ranger RussetProtoplasts from leaves on 3-week-old potato plants (Ranger Russet) were isolated and transformed with plasmids encoding TALEN pairs. Following transformation, exon 1 was amplified by PCR and mutations were assessed by 454 pyro-sequencing. Percentage of vacuolar invertase sequences containing TALEN-induced mutations. Total refers to the combined percentage of mutations in all four alleles. Else refers to sequences that could not be assigned an allele type due to TALEN-induced mutations that removed the allele-defining single nucleotide polymorphisms.

22 Recovery of potato lines carrying mutations within vacuolar invertase (VInv)Approach and timeline to regenerate plants with mutations in VInv. Protoplasts were transformed with plasmids encoding the VInv_T2 transcription activator-like effector nucleases pair and were cultured in nonselective regeneration medium. Following shoot and root formation, potato plantlets were transferred to soil. Examples of plant lines carrying mutations in one or more of the VInv alleles. WT, wild type.

23 Quality assessment of mutant potato linesAnalysis of sugar content within potato tubers stored at 4 °C for 14 days. Error bars represent standard deviation. Analysis of acrylamide content in potato chips that were processed from tubers that were stored at 4 °C for 14 days. Images of potato chips after being processed from tubers stored at 4 °C for 14 days. The colorimetric score is listed to the right of the image.

24 Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System Nathaniel M. Butler 1 , Paul A. Atkins 2 , Daniel F. Voytas 2 , David S. Douches 1 * (Published: December 14, 2015) This report demonstrates the use of CRISPR/Cas for targeted mutagenesis in both diploid and tetraploid potato. CRISPR/Cas reagents targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were expressed in leaf explants via Agrobacterium tumefaciens (Agrobacterium) using a conventional 35S T-DNA expression vector [18] or a modified geminivirus T-DNA expression vector [19]. Both sgRNAs and T-DNAs tested were capable of generating targeted mutations in stable events. Single targeted mutations in primary events were capable of being carried through clonal generations and the germline as Cas9-free progeny. The tetraploid S. tuberosum cultivar “Désirée” (Désirée) and a diploid selfincompatible breeding line, MSX (X914-10) were used in the study. X was produced from a cross between the doubled-monoploid (DM) S. tuberosum Group Phureja line used to construct the potato reference genome [20] and 84SD22, a heterozygous S. tuberosum x S. chacoense hybrid breeding line and has high transformation efficiency [21]. Fig 1. Generation of targeted mutations in callus tissues of potato using CRISPR/Cas reagents. A. Target sites of single-guide RNA within potato StALS1 and -2 genes. A single nucleotide polymorphism (lowercase) exists in the gRNA746 target site of StALS2 but not gRNA751. AloI and BslI restriction enzyme sites exist in sgRNA target sites of both genes (underlined). Arrows indicate primers used for enrichment PCR and restriction enzyme digestion assays. PAM sequences are in gray. B. Modified enrichment PCR assay using potato callus tissue transformed with gRNA746 and gRNA751 CRISPR/Cas.

25 Fig 1. Generation of targeted mutations in callus tissues of potato using CRISPR/Cas reagents. A. Target sites of single-guide RNA within potato StALS1 and -2 genes. A single nucleotide polymorphism (lowercase) exists in the gRNA746 target site of StALS2 but not gRNA751. AloI and BslI restriction enzyme sites exist in sgRNA target sites of both genes (underlined). Arrows indicate primers used for enrichment PCR and restriction enzyme digestion assays. PAM sequences are in gray. B. Modified enrichment PCR assay using potato callus tissue transformed with gRNA746 and gRNA751 CRISPR/Cas reagents. Total genomic DNA was subjected to PCR amplification of the StALS target site (bottom image; 448 bp), digested overnight with AloI (lanes 1, 3, 5, 7, 9, 11) or BslI (lanes 2, 4, 6, 8, 10, 12), and reamplified (top image; 448 bp) to generate an enriched amplicon. Enriched band intensities were normalized by dividing the quantified band intensity of the enriched band by the primary PCR amplicon (S1 Table). Positive (+), negative (-) and non-detectable (ND) enriched bands have normalized intensities equal or over 0.5, less than 0.5 and equal or more than 0.05, or less then 0.05, respectively. Diploid (X; lanes 1–6) and tetraploid (D; lanes 7–12) genotypes were tested using both sgRNAs in the conventional 35S (M; lanes 1, 2, 7, 8) and geminivirus LSL (L; lanes 3, 4, 9, 10) T-DNA backbones. Wild-type (wt; lanes 5, 6, 11, 12) genomic DNA was used as non-transformed controls.

26 Fig 2. Generation and cloning of targeted mutations in primary events of potato using CRISPR/Cas reagents. A. Restriction enzyme digestion assay of diploid (X; lanes 2–4) and tetraploid (D; lanes 5–8) primary events. Total genomic DNA from primary events was subjected to PCR amplification of the StALS target site and digested overnight with AloI yielding a 448 bp resistant band and 326 bp and 122 bp digested bands. Wild-type X (WT; lane 1) and Désirée (Fig 3A) genomic DNA were used as a negative controls. B. Cloned targeted mutations in primary events of potato. Diploid (X) and tetraploid (D) events constitutively expressing gRNA746 (46) and gRNA751 (51) CRISPR/Cas reagents were used for cloning. Resistant bands from restriction enzyme digestion assays were excised from 2.0% agarose gels, purified, and subcloned for Sanger sequencing. Sanger reads from each event were aligned to StALS1 and -2 wild-type sequence (WT) from each sgRNA target site (gRNA746; top alignments, gRNA751; bottom alignments). The lengths of deletions (-) or insertions (+) are in parenthesis to the left of each cloned mutation and the number of reads generated in the primary event (T 0 ) or first clonal generation (CG 1 ) are in brackets on the right. All targeted mutations were cloned from StALS1 unless indicated on the right. PAM sequences are in gray.

27 Технологии CRISPR / Cas и сайт-специфические нуклеазы (SSN) позволяют проводить направленный мутагенеза генов-мишеней и непосредственно/напрямую определять функции генов.

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