Abstract
This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.
Highlights
Restriction endonucleases were discovered originally as a bacterial defensive mechanism against the invading phages or plasmid DNA in 1970 [1]
This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity
Plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA
Summary
Restriction endonucleases were discovered originally as a bacterial defensive mechanism (restriction-modification system) against the invading phages or plasmid DNA in 1970 [1]. The in vitro reconstitution experiments for double-stranded DNA cleavage required the Cas enzyme, spacer 2 sequence with functional protospacer adjacent motif (PAM), 42-nucleotide CRISPR-derived RNA (crRNA) containing a spacer 2 sequence, and 75-nucleotide trans-activating crRNA (trancrRNA) [6]. Native gel mobility assay was used to determine the binding affinity of complex for target sequence Both G nucleotides of the PAM are required for the affinity of Cas9-tracrRNA:crRNA to the target DNA and the subsequent DNA cleavage. In type II of class 2 systems including Cas, crRNA and a trans-activating antisense RNA (tracrRNA) forms a RNA duplex base-paired through the complementary sequence to CRISPR repeats [11]. Matching PAM and the close proximal seed sequence is a crucial quality control step that is required for the complete displacement of the non-complementary strand of the target DNA by the crRNA guide forming R-loop conformation [7]. This allows the Cas9-RNA duplex to probe the identity of the nucleotides of the seed sequence, driving further step-wise destabilization of DNA duplex
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