crisprcas9.ppt

CRISPR/cas9 Molecular Biology First of all:What is the CRISPR/cas9? CRISPR/Cas systems are used by various bacteria and archaea to mediate defense against viruses and other foreign nucleic acid. Recent work has shown that Type II CRISPR/Cas systems can be engineered to direct targeted double-stranded DNA breaks in vitro to specific sequences by using a single guide RNA with complementarity to the DNA target site and a Cas9 nuclease (Jinek et al., Science 2012). Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to memorize and later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA.Cas9 performs Secondly:CRISPR/cas9 is a genetic tool. The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and TALEN proteins, Cas9 is becoming a prominent tool in the field of genome editing. Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA.Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and Zinc-fingers),in addition engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. While native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide - the CRIS