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Crick Lecture- The transformative CRISPR-Cas9 genome engineering technology: lessons learned from bacteria

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The RNA -programmable CRISPR -Cas9 system has recently emerged as a transformative technology in biological sciences, allowing rapid and efficient targeted genome editing, chromosomal marking and gene regulation in a large variety of cells and organisms. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants are programmed with single guide RNAs (sgRNAs) to target site-specifically any DNA sequence of interest given the presence of a short sequence (Protospacer Adjacent Motif, PAM ) juxtaposed to the complementary region between the sgRNA and target DNA .

Originally, CRISPR -Cas is an RNA -mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements. Short crRNA (CRISPR RNA ) molecules containing unique genome-targeting spacers guide Cas protein(s) to invading cognate nucleic acids to affect their maintenance. CRISPR -Cas has been classified into three main types and further subtypes. CRISPR -Cas9 originates from the type II system that has evolved unique molecular mechanisms for maturation of crRNAs and targeting of invading DNA , which my laboratory has identified in the human pathogen Streptococcus pyogenes. During the step of crRNA biogenesis, a unique CRISPR -associated RNA , tracrRNA, base pairs with the repeats of precursor-crRNA to form anti-repeat-repeat dual-RNAs that are cleaved by RNase III in the presence of Cas9, generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual-tracrRNA-crRNAs guide Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that Cas9 can be programmed with sgRNAs mimicking the natural dual-tracrRNA-crRNAs to target site-specifically any DNA sequence of interest. I will discuss the biological roles of CRISPR -Cas9, the mechanisms involved, the evolution of type II CRISPR -Cas components in bacteria and the applications of CRISPR -Cas9 as a novel genome engineering technology.

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