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CRISPR‐Cas9: Power And Challenges
Author(s) -
Charpentier Emmanuelle
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.118.1
Subject(s) - trans activating crrna , crispr , cas9 , biology , genome editing , rna , computational biology , genetics , dna , crispr interference , guide rna , plasmid , gene
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. The system is efficient, versatile and easily programmable. Originally, CRISPR‐Cas is an RNA‐mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements (phages, plasmids). Short crRNA (CRISPR RNA) molecules containing unique genome‐targeting spacers commonly 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 CRISPR‐Cas 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 (formerly Csn1), generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual‐tracrRNA‐crRNAs guide the endonuclease Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that the endonuclease 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.