Addgene_ CRISPR Guide.pdf - Addgene CRISPR Guide This...

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1/10/2020 Addgene: CRISPR Guide 1/18 This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies. Close CRISPR Guide CRISPR Overview Class 2 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems, which form an adaptive immune system in bacteria, have been modi±ed for genome engineering . Prior to CRISPR, genome engineering approaches like zinc ±nger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs) required scientists to design and generate a new nuclease pair for every genomic target. Due to its comparative simplicity and adaptability, CRISPR has rapidly become the most popular genome engineering approach. Engineered CRISPR systems contain two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-de±ned 20 nucleotide spacer that de±nes the genomic target to be modi±ed. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA. CRISPR was originally employed to knock out target genes in various cell types and organisms, but modi±cations to various Cas enzymes have extended CRISPR to selectively activate/repress target genes, purify speci±c regions of DNA, image DNA in live cells, and precisely edit DNA and RNA. Furthermore, the ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies. This advantage makes CRISPR perfect for genome-wide screens. This guide will provide a basic understanding of CRISPR biology, introduce the various applications of CRISPR, and help you get started using CRISPR in your own research. Generating a Knockout Using CRISPR
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1/10/2020 Addgene: CRISPR Guide 2/18 You can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cas12a (also known as Cpf1) and a gRNA speci±c to the targeted gene. The genomic target can be any 20 nucleotide DNA sequence, provided it meets two conditions: 1. The sequence is unique compared to the rest of the genome. 2. The target is present immediately adjacent to a Protospacer Adjacent Motif (PAM). The PAM sequence serves as a binding signal for Cas9, but the exact sequence depends on which Cas protein you use. We'll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences . Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9.
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  • Fall '19
  • DNA, gRNAs, Addgene

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