Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

Abstract

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a doublestrand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate sitespecific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and proteinmediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.nuclease | site-directed mutagenesis | RNA interference | DNA interference

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…Moreover, instead of integrating into the host genome, lentiviruses can transiently present in a circular double-stranded format in the host cells. The CRISPR/CRISPR-associated genes (Cas) system has been reported to functionally inactivate invading plasmids in bacterial cells 10 ; we therefore examined whether the CRISPR/ Cas9 system can also be used to inactivate circular foreign plasmids (double-stranded circular DNA) in human cells. By using the same backbone of EGFP reporter in previous lentivirus assays ( Supplementary Fig.…”

“…The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is known to play a major role in the adaptive immune response to foreign plasmids and viruses in about 40% of bacteria [9][10][11] . Recently, several research groups have successfully applied the type II CRISPR systemSpCas9 protein from Streptococcus pyogenes with guided RNA (gRNA)-for targeted genome editing in diverse cell types and organisms, including human cells [12][13][14] .…”

Generation of gene-modified mice via Cas9/RNA-mediated gene targeting

“…These two RNA elements form a crRNA:tracrRNA duplex that directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. The 3 nucleotides (nt) located immediately at the 3′ side next to the protospacer sequence constitute the protospacer adjacent motif (PAM) that is required to ensure the cleavage specificity in target sequences [9,10].…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…Depending on the architecture of the effector-CRISPR RNA (crRNA) interference module, different CRISPR-Cas systems could be assigned into two classes [1]: class 1 systems (multi-subunit complex, such as Cascade) [4,5] and class 2 systems (single enzyme, such as Cas9) [6,7]. Cas9 is the signature member of class 2 systems, which functions as a multi-domain endonuclease, along with crRNA and trans-activating crRNA (tracrRNA), or alternatively with a synthetic single-guide RNA (sgRNA), to cleave both strands of the target DNA [6][7][8]. A short and conserved protospacer adjacent motif (PAM) sequence near the target site is required for the cleavage process of Cas9 [9,10].…”