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Nuclease domain consists of type II restriction endonuclease Fok1.

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HNH endonuclease domain; 42 nt long gRNARequire one RNA (crRNA); Produce staggered cut ends; easier to deliver in low capacity vectors ex.

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Major differences between CRISPR classes are based on the composition of crRNP complexes.

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Adaptation involves the invading DNA from virus or plasmids that are cleaved into small fragments and incorporated into CRISPR locus.

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Cas9 is a huge protein possessing multiple domains (RuvC domain at the amino terminus and the HNH nuclease domain positioned in middle) and two small RNAs namely crRNA and tracrRNA.

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These sticky ends would provide more efficient genomic insertions due to sequence complementarity into a genome.

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Creating variations in the gene pool is the foremost requirement for developing novel plant varieties.

Class 1 CRISPRs have multiple subunit effector complexes while class 2 CRISPRs concentrates most of their functions with single protein effectors.

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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

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Cas9 systems has broadened the application of this technology manifold.

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RNA polymerase III promoters such as AtU6, TaU6 etc.

Gene editing uses engineered SSNs to delete, insert or replace a DNA sequence.

HEPN domains that are biochemically characterized as ssRNA specific endoribonucleases but there is a significant line of difference between these two types.

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They also noted that spacers share a common end sequence, now known as PAM.

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Table 4Different plasmids with specific Cas9 activity.

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The choice of the vectors largely depends upon the type of the expression system to be worked on, type of restriction sites present to insert sgRNA and the type of Cas9 system.

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Cas9: Cas9 endonuclease is guided by sgRNA (single guide RNA: crRNA and tracrRNA) for target specific cleavage.

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Cas9 genomes editing in plants require suitable vector system (codon optimized Cas9 gene and promoters for Cas9 and sgRNA), efficient target sites and transformation method used in appropriate plant species.

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In the presence of donor DNA digested with the same endonuclease leaving behind similar overhangs, HR can be achieved leading to gene modification and insertion.

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