Crisper uses

Uses of CRISPER for crop improvement

1. Novel CRISPR/Cas applications in plants: Genome editing and beyond Akshay Sakhare Sudhir Kumar

2. Genome editing Targeted genome editing is a broadly applicable approach for efficiently modifying essentially any sequence of interest in living cells or organisms. First generation genome editing technologies uses meganucleases , ZFNs and TALENs which relies on the use of engineered nucleases and involve tedious procedures to achieve target specificity, are labor intensive and time-consuming. These are basically artificial proteins composed of a customizable sequence-specific DNA-binding domain fused to a nuclease that cleaves DNA in a non-sequence-specific manner. Genome editing with engineered nucleases was named the year 2011 ‘ Method of the Year ’ . In contrast, second generation genome editing techniques including CRISPR/Cas9 involve easier design and execution methodologies that are also more time- and cost-effective.

3. Major tools for Targeted Genome Editing

4. ZFNs and TALENs Mr tools for TGE A large number of zinc-finger arrays have been fused to a nonspecific nuclease domain from the Fok I restriction enzyme to create zinc-finger nucleases (ZFNs) TALENs are similar to ZFNs and comprise a nonspecific Fok I nuclease domain fused to a customizable DNA-binding domain. DNA-binding domain is composed of highly conserved repeats derived from transcription activator-like effectors (TALEs) Nature Reviews Molecular Cell Biology doi:10.1038/nrm3486

5. CRISPR: Hall mark of acquired immunity in Bacteria CRISPR Abundancy 50% Bacterial taxa 90% archeal taxa Spacer Seq . CRISPER array Metagenomic sequencing of many bacterial genome revealed “A short palindromic repeats” with highly variable intervening seq. Later these arrays were mapped with some of phage genome infecting frequently. Along with these CRISPR loci , a protein encoding gene Cas9 is present upstream and reported in almost all loci across the several bacterial genome. Bolotin et.al, 2005;Mojica et.al, 2005

6. Acquire immunity in Bacteria Wiedenheft et.al 2012 Cas complex mediate cleavage of protospacer sequence of phage DNA And subsequent acquisition of Spacer seq. to the 5’ end of the CRISPR Loci repeat–spacer–repeat architecture

7. crRNA biogenesis Interference Wiedenheft et.al 2012 Acquire immunity in Bacteria

8. Bortesi and fisher,2015 In the native system CRISPR RNA Determine target specificity tracrRNA Stablizes the structure & Activates Cas9 for cleavage Protospacer Adjacent Motif (PAM) 3 nt NGG/NAG downstream to target is prerequisite for Cleavage by Cas9 Seed Sequence A part of trget specificity determing crRNA required for Efficient Binding In the Engineered system Cas9 All Cas9 protein has two domain HNH domain: cleaves strand of Dna complementary to gRNA RuvC domain: cleaves non-complementary strand of DNA All Cas9 protein are arginine rich which might helps in negatively charged Nucleic acid binding single guide RNA molecule A chimera generated by fusing the 3′ end of the crRNA to the 5′ end of the tracrRNA . So, Can be reprogrammed using single gRNA RNA-guided DNA Cleavage by Cas9

9. HDR It is a recombinational repair mechanism of DSB. Gene Fixing at desired site can be achieved if Donor DNA with Homology to seq. being cleaved by Cas9 is provided. Gene insertion and Gene modification can be achieved. NHEJ Emergency repair mechanism of DSB usually causes insertion or Deletion of Few nucleotides  Frame shift mutation  Gene Knock out. If Donor DNA with compatible ends are present, then gene insertion by NHEJ is possible Duodna et.al , 2012 A crisper way to fix genes at desired position

10. Wiedenheft et.al 2012 In type I and III systems, a CRISPR-specific endoribonuclease cleaves 8 nucleotides upstream of each spacer sequence. In type III systems, the repeat sequence on the 3ʹ end of the crRNA is trimmed by an unknown mechanism. A trans -acting antisense RNA ( tracrRNA ) with complementarity to the CRISPR RNA repeat sequence forms an RNA duplex that is recognized and cleaved by cellular RNase III This cleavage intermediate is further processed at the 5ʹ end resulting in a mature, approximately 40-nucleotide crRNA with an approximately 20-nucleotide 3ʹ-handle. Diversity of CRISPR system Duodna et.al, 2012

11. Classification within the context of site-directed nuclease (SDN) technology Massel et al, 2021

12. Cas9 orthologues from bacterial species show differences in their PAM repertoire Jaganathan et al,2018

13. Beyond Genome Editing Bortesi and fisher, 2015.

14. It is now possible to induce changes in plant genomes from a single nucleotide to the restructuring of whole chromosomes on the Mb scale Huang, T. K., & Puchta , H. 2021.

15. Hypothetical pathway of C-to-G base transversion by using the BE technology Huang, T. K., & Puchta , H. 2021. Yarra , R., & Sahoo , L. 2021.

16. Prime editing techniques Huang, T. K., & Puchta , H. 2021.

17. Chromosome engineering Huang, T. K., & Puchta , H. 2021.

18. Advantages of CRISPR/cas9 System Very high efficiency No drug selection required Rapid construction and easy delivery Efficient biallelic targeting Multiplexing possible Successful in different cell types and species.

19. Applications and implications in plant improvement Precise and predictable genome editing To eliminate genes that hampers food quality Confers susceptibility To divert metabolic flux for valuable end products Deletion of genes Insertion of genes Molecular Stacking Desirable gene Stacking at single locus can be mobilized to elite germplasm There is hope and confidence that plants altered by the excision of a few nucleotides using genome editing tools such as CRISPR/Cas9 would not be classified as genetically modified organisms. ( Hartung and Schiemann, 2014; Li et al., 2012; Podevin et al., 2013). Metabolic engineering & Molecular farming

20. Application of CRISPR/ Cas genome editing in gene functional study Zhang, Dangquan , et al, 2020

21. Rapid and transgene-free development of abiotic stress-tolerant crops using the multiplex CRISPR/Cas9 technique in comparison with conventional mutation breeding techniques Zafar et al, 2020 .

22. Challanges Off-target mutations PAM dependence gRNA production and designing Delivery methods

23. Strategies to increase target specificity Bortesi and fisher, 2015.

24. http://dx.doi.org/10.1016/j.copbio.2014.11.007 Working with CRISPR/Cas9 system

25. System construction and expected outcomes of the approach

26. SlHyPRP1 indel alleles obtained from the multiplexed editing

27. Performance of the edited lines at the germination stage under salinity stress (150 mM NaCl ).

28. Salinity tolerance at the germination stage, growth stage and Performance of the edited lines at the vegetative growth stage under salinity stress

29. Conclusion This study revealed variable roles of the SlHyPRP1 domains in salt stress responses and indicated that precise removal of each of the domains generated salt stress-tolerant events in tomato. Precise elimination of SlHyPRP1 negative-response domain(s) has led to high salinity tolerance (up to 150 mM NaCl ) in this experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multi domain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

30. Representative dst Indel mutations of alleles identified from sequence analysis of PCR amplicons from dst mutants The dst∆184–305 mutation confers enhanced flag leaf area. a and b showing Flag Leaf width while c shows Flag leaf area of WT and dst∆184–305 mutant

31. Stomatal density and expression analysis of genes involved in stomatal development .

32. Chlorophyll retention assay of dstD184–305 mutant

33. Drought and Salt tolerance of dstD184–305 mutant