Lecture 15 – CRISPR-Cas9 and RNAi PDF

MIC115 Recombinant DNA Cloning 24FQ Lecture 15 – CRISPR-Cas9 and RNAi Tools for functional studies Text with gray bars: you do not need to remember SUGGESTED VIDEOS Method of the Year...

MIC115 Recombinant DNA Cloning 24FQ Lecture 15 – CRISPR-Cas9 and RNAi Tools for functional studies Text with gray bars: you do not need to remember SUGGESTED VIDEOS Method of the Year 2011: Gene-editing nucleases - by Nature Video (TALEN) https://www.youtube.com/watch?v=zDkUFzZoQAs Bacterial Adaptive Immunity with CRISPR/Cas9 https://www.youtube.com/watch?v=MbJ7Hnc2K3Q Gene Silencing Methods: CRISPR vs. TALENs vs. RNAi https://www.youtube.com/watch?v=U3Z4u0DKbx0&t=352s LECTURE 15 TOPICS History: Site-directed DSBs CRISPR-mediated "adaptive immunity" in bacteria CRISPR-mediated genome editing Other CRISPR applications RNA interference (comparison with CRISPR) LECTURE 15 LEARNING GOALS Understand the CRISPR-mediated "adaptive immunity" in bacteria Describe how CRISPR-mediated genome editing works Understand the outline of RNA interference Compare basic experimental steps of CRISPR and RNAi I. What can we do with genome editing? Animal models of human diseases Study gene functions in cells Infection-resistant crops Algae to produce fat for biofuels Hold promise to precisely correct mutations in patients II. History: Site-directed DSBs Zinc Finger Nuclease (ZFN) 2000s Zinc Finger domains recognized 3 bases each Nuclease domain introduces nicks, design both sides to make nicks to produce DSBs Not commonly used anymore 1 MIC115 Recombinant DNA Cloning 24FQ Transcription Activator-Like Effector Nucleases (TALEN) 2011〜 TAL effectors recognize each base Nuclease domain introduces nicks, design both sides to make nicks to produce DSBs Very specific, but not commonly used anymore Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 2012〜 III. CRISPR-mediated "adaptive immunity" in bacteria The CRISPR system is an important bacterial defense system against invading phage (or conjugative plasmids). It allows bacteria to remember invading phages and destroy phages. (Keep phage-derived DNA in the genome and use them to destroy phages) The system is similar in principle to adaptive immunity in mammals. Upon phage infection: A new spacer derived from the invasive phage DNA is incorporated into the CRISPR array by the acquisition machinery (Cas1, Cas2, and Csn2 proteins). These spacers are segregated by direct repeats in between. The spacers are transcribed into a long precursor CRISPR RNA (pre-crRNA). The trans-activating CRISPR RNA (tracrRNA) is transcribed. It hybridizes with the pre- crRNA direct repeats, forming an RNA duplex that is processed by host endogenous RNase III and other nucleases. The mature crRNA–tracrRNA structure engages Cas9 endonuclease and further directs it to cleave foreign DNA containing a 20-nt crRNA complementary sequence preceding the PAM sequence. Just FYI: https://www.annualreviews.org/doi/10.1146/annurev-biophys-062215-010822 Figure 1 CRISPR–Cas9-mediated DNA interference in bacterial adaptive immunity. (a) A typical CRISPR locus in a type II CRISPR–Cas system comprises an array of repetitive sequences (repeats, brown diamonds) interspaced by short stretches of nonrepetitive sequences (spacers, colored boxes), as well as a set of CRISPR-associated (cas) genes (colored arrows). Preceding the cas operon is the trans-activating CRISPR RNA (tracrRNA) gene, which encodes a unique noncoding RNA with homology to the repeat sequences. Upon phage infection, a new spacer (dark green) derived from the invasive genetic elements is incorporated into the CRISPR array by the acquisition machinery (Cas1, Cas2, and Csn2). Once integrated, the new spacer is cotranscribed with all other spacers into a long precursor CRISPR RNA (pre-crRNA) containing repeats (brown lines) and spacers (dark green, blue, light green, and yellow lines). The tracrRNA is transcribed separately and then anneals to the pre-crRNA repeats for crRNA maturation by RNase III cleavage. Further trimming of the 5′ end of the crRNA (gray arrowheads) by unknown nucleases reduces the length of the guide sequence to 20 nt. During interference, the mature crRNA–tracrRNA structure engages Cas9 endonuclease and further directs it to cleave foreign DNA containing a 20-nt crRNA complementary sequence preceding the PAM sequence. (b) 2 MIC115 Recombinant DNA Cloning 24FQ Schematic representation of the domain organization of the representative Cas9 orthologs from distinct subtypes. Asterisks denote conserved, key residues for Cas9-mediated DNA cleavage activity. Abbreviations: Arg, arginine-rich bridge helix; crRNA, CRISPR RNA; CTD, C- terminal domain; nt, nucleotide; NUC, nuclease lobe; PAM, protospacer adjacent motif; REC, recognition lobe; tracrRNA, trans-activating CRISPR RNA. Single-guide RNA (sgRNA) was developed to mimic the mature crRNA–tracrRNA structure. This single RNA sequence is sufficient for CRISPR experiments. Simplified CRISPR/Cas9 nuclease system: 1. The Cas9 nuclease 2. Single guide RNA (sgRNA=crRNA+tracrRNA) The CRISPR-Cas9 System can be directed to cleave an arbitrary, user-defined DNA sequence III. CRISPR experiments in cultured cells. sgRNA- and Cas9-containing plasmids can be transfected together to cultured cells. Alternatively, synthesized sgRNA and a Cas9-containing plasmid can be transfected together to cultured cells. Quiz: how can we introduce these molecules to cells? A. Plasmids can be introduced with lipofection or lentiviral transduction. Synthesized sgRNA can be introduced with lipofection. Quiz: how about mouse fertilized eggs? A. Microinjection Expression of sgRNA and Cas9 and in cultured cells. After transcription of plasmids, Cas9- sgRNA complex formation takes place. Then, the Cas9-sgRNA complex generates a double-strand break at the target site. IV, Other CRISPR applications Catalytically inactive “dead” Cas9 (dCas9): no-nuclease activity but binds to DNA Fusion protein with dCAS9 and an effector protein can be engineered. Fusion protein with dCAS9 and an effector protein (activator) can be used for gene activation (CRISPR activation: CRISPRa). Fusion protein with dCAS9 and an effector protein (repressor) can be used for gene repression (CRISPR interference: CRISPRi). Let’s challenge: What effector would you be interested in? 3 MIC115 Recombinant DNA Cloning 24FQ We can target any proteins to specific sites (GFP, etc). V. RNA interference (RNAi) RNA interference (RNAi): RNA silencing: “knockdown” siRNA (21- to 23-nucleotide double-stranded RNAs) can trigger RNAi siRNA works with a protein complex called “RNA-induced silencing complex (RISC)” Degrading mRNA transcript (low expression: not zero) with perfect match: RNA cleavage Blocking translation with some miss matches Structure of siRNA Two RNA strands form a duplex 21 bp long with 3' dinucleotide overhangs on each strand. The siRNA mechanism is similar to the endogenous microRNA (miRNA) pathway that suppress target genes RNA-induced silencing complex (RISC) contains Argonaute (AGO): endonuclease. VI. RNA interference (RNAi) experiments Introduce “synthesized siRNA” or “Short hairpin RNA (shRNA)-containing vector:a siRNA precursor can be expressed from a vector” to cultured cells. Short hairpin RNA (shRNA) can be process to siRNA by a nuclease, DICER. “synthesized siRNA” can be introduced by direct transfection by lipid-based reagents (Lipofection) “Short hairpin RNA (shRNA)-containing vector:a siRNA precursor can be expressed from a vector” can be introduced by transduction with lentiviral vectors 4