Lecture 15 (RNAi, CRISPR).pdf

BIOL 366 – lecture 15 Topics: ▪ RNA interference ▪ CRISPR/Cas gene editing system Text Section: 21.1 – 2.13 & 22.4 Articles: 1) Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Science 327: 167-170. 2) The Origene Crispr Manual Key terms: microRNA (miRNA): short i...

BIOL 366 – lecture 15 Topics: ▪ RNA interference ▪ CRISPR/Cas gene editing system Text Section: 21.1 – 2.13 & 22.4 Articles: 1) Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Science 327: 167-170. 2) The Origene Crispr Manual Key terms: microRNA (miRNA): short interfering RNA (siRNA), RNA interference (RNAi), antisense-mediated gene silencing, Dicer, RNA-induced silencing complex, Argonaute, Drosha, Lentivirus, CRISPR-Cas System, Cas genes, Cas proteins, crRNA, gRNA (guide RNA), CRISPR-Cas System, Cas genes, Cas proteins, crRNA, gRNA (guide RNA), codon optimization, Yeast 2 hybrid system 1 Gene silencing In the 1980s and 1990s: Attempts to reduce gene expression via antisense mRNA (complementary to the sense mRNA) to block protein expression. Rationale: Base pairing between the antisense RNA and the target mRNA would prevent translation, lead to mRNA degradation, or both. 2 Mechanism of gene silencing via antisense - A copy of the target gene is Inserted in the genome in reverse orientation - The gene produces sense mRNA; the inverted gene produces antisense mRNA - The sense mRNA and the antisense mRNA base pair - Translation is impeded - Duplex mRNA is degraded Promoter: Gene: 5’------ATGAAAAAAGGGTTA-------------TAACCCTTTTTTCAT-------------3’ 3’------TACTTTTTTCCCAAT-------------ATTGGGAAAAAAGTA-------------5’ Transcription 5’augaaaaaaggguua3’ 5’uaacccuuuuuucau3’ 5’augaaaaaaggguua 3’ 3’uacuuuuuucccaau 5’ mRNA sequences 3 Gene silencing However, in plants: - Antisense RNA worked reasonably well, but failed to efficiently (>90%) suppress gene expression in many cases - Expression of “sense” RNA was often more effective in suppression of the targeted gene thank antisense. This was called cosuppression. - Researchers subsequently demonstrated in plants, worms, and other eukaryotes that cosuppression is mediated by short interfering RNA molecules (siRNAs). 4 RNA interference (RNAi) 5 RNA interference (RNAi): • A method of gene silencing that prevents mRNA translation, usually by targeting it for degradation • Can be mediated by siRNA or microRNA (miRNA) o miRNA is endogenous to the cell, produced by nucleases from longer transcripts encoded in the genome o siRNAs are: ➢ Generated by the same cellular machinery that produce miRNA ➢ Introduced into the cell by viral infection or experimental manipulation 6 RNA interference (RNAi) microRNA (miRNA): - small single-stranded RNA (21 to 23 nucleotides, after processing) - involved in natural gene silencing by: - inhibiting translation - promoting the degradation of particular mRNAs short interfering RNA (siRNA): - a short (~21 to 27 nucleotide) single-stranded RNA - created from exogenous DNA/RNA introduced by a researcher - participates in the RNAi gene silencing (as for miRNA) 7 Role of RNA Interference in nature In nature, RNAi and related pathways: - Control developmental timing in some organisms (e.g., nematode C. elegans) - Protect against invading RNA viruses (especially important in plants, which lack an immune system) - Control the activity of transposons - Small RNA molecules also play a critical, although still undefined, role in the formation of heterochromatin. 8 Mechanism of RNAi action 9 miRNA synthesis and action Summary i) miRNAs are transcribed by RNA Polymerase II / Pol III as pri-miRNAs (primary miRNA transcripts). ii) Pri-miRNAs are processed to produce mature miRNA. iv) Mature miRNA molecules bind and promote degradation of the target mRNA 10 miRNA production and processing Rles of Drosha and Dicer: (By RNA Pol II / III) - Unique ribonucleases - Produce siRNA from a pri- & pre-miRNA - Drosha functions in the nucleus - Dicer functions in the cytosol as part of RISC (RNA-induced silencing complex) FIGURE 22-16 11 miRNAs of eukaryotic genomes target mRNAs for gene silencing Fig. 22-16 12 A few notes on RNAi. The structure of RNAi gene and transcript - A portion of the pre-miRNA is identical to your mRNA target (the mRNA to be degraded), so that the target mRNA can be identified. This is yellow in the Fig. - A second portion of the pre-siRNA is different from the mRNA so it can form a loop – Also yellow in Fig. - A third portion of the pre-miRNA is complementary to your mRNA target (the mRNA to be degraded). This is purple in the Fig. 13 RNAi is a powerful tool in molecular biology RNA Interference in Biotechnology Fig. 22-14 silencing of genes for flower pigmentation by RNAi in transgenic petunia Flower from wild type plant flowers from genetically modified plants 14 siRNA In addition to pre-miRNA, Dicer can also recognize and cleave EXTERNALLY supplied dsRNAs to produce siRNA - dsRNA could be introduced by experimenters (biotechnology) - dsRNA could be introduced by natural sources such as viruses So: - miRNA is a natural way of regulating gene expression in an organism. The miRNA genes are within the organism’s genome. - siRNA is introduced into the cell from external sources. - siRNA is controlled by the same cellular machinery involved in miRNA action. 15 siRNA • Diced products are 21 to 27 bp in length (siRNAs) • siRNAs regulate gene expression by mechanisms similar to those described for miRNAs. • In the cytoplasm, siRNAs can form perfect or imperfect base pairings with targeted mRNA. • Perfect base pairing between the siRNA and its target triggers degradation of the targeted RNA through the normal pathways • Imperfect base pairing between the siRNA and its target leads to impeding of translation, and eventual transcript degradation (Fig 22-17) 16 The RNA-dependent RNA Polymerases (rdRP) can amplify the siRNA in nematodes (not all organisms) • The siRNAs targets an mRNA • The bound siRNA serves as a primer for RdRP • RdRP creates a longer doublestranded RNA • dsRNA is used as a source of more siRNAs by Dicer • Results in stronger gene silencing (Fig 22-18) 17 RNAi in silencing genes in human cells (see next slide too) • RNA homologous to the target gene is cloned into a lentiviral vector • The recombinant virus is used to infect human cells • In the host cell, viral RNA is reverse transcribed to duplex DNA, which is integrated into the host cell genome. • Transcription of the transgene produces siRNA specific to the target gene Note: RNA should be able to form shorthairpin RNA (shRNA). See next slide. 18 RNAi in silencing genes in human cells One way to induce RNAi is to introduce a short-hairpin RNA (shRNA) in the target cell. To do this: i) Design an RNA molecule that can form shRNA. The molecule should be identical/homologous to the target gene. Example: (5’acguccuagacccgcNNNNNNNNNgcgggucuaggacgu3’) Stem: ~ 10 – 27 (ideal 20 – 25) pairs Loop length: ~ 7 – 20 pairs ii) Insert the RNA molecule in a lentivirus vector iii) Transfect cells with the virus 19 Gene editing The CRISPR / Cas system 20 The CRISPR / Cas system A “nuclease-RNA” complex that cuts DNA in specific location • CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats • Cas: CRISPR-Associated protein 21 The CRISPR/Cas system in Nature • CRISPR loci typically consist of several noncontiguous direct repeats separated by stretches of variable sequences called spacers • CRISPR loci are often adjacent to Cas genes (CRISPR-associated) • Cas genes encode a large and heterogeneous family of proteins that carry functional domains typical of nucleases, helicases, polymerases, and polynucleotide-binding proteins. • In nature, the CRISPR/Cas system provides bacteria with immunity against invading DNA (e.g., bacteriophage, plasmid) Fig 1, from Horvath and Barrangou (2010). One of the 4 CRISPR/Cas systems present in Streptococcus thermophilus Reference: Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Science 327: 167-170. 22 Overview of the CRISPR/Cas mechanism of action. (A) Immunization process Incoming foreign DNA (viruses, plasmids) Cas complex recognizes foreign DNA Integrates a novel repeat-spacer unit at the leader end of the CRISPR locus (Fig 2, Horvath and Barrangou (2010)) Reference: Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of 23 Bacteria and Archaea Science 327: 167-170. Overview of the CRISPR/Cas mechanism of action. (B) Immunity process Terms: • The CRISPR repeat-spacer array is transcribed into precrRNA, and processed into crRNAs. • The crRNA is used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid. Overview of the CRISPR/Cas mechanism of action. (Fig 2, Horvath and Barrangou (2010)) Reference: Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Science 327: 167-170. 24 Cleavage site by Cas9 Reference: Bannikov and Lavrov 2017. CRISPR/CAS9, the King of Genome Editing Tools Molecular Biology, 2017, Vol. 51, No. 4, pp. 514–525. 25 Precision genetic engineering using the CRISPR/Cas system using the Origene Crispr/Cas system. (See Origene Crispr Manual https://www.origene.com/ ) See a number of videos here: https://www.origene.com/support/learningresources/videos-webinars Watch: https://www.youtube.com/watch?v=0dRT7slyGhs&feature=youtu.be And https://www.youtube.com/watch?v=MzMsgmeEOhI&list=PL4_fJegcjcJ_doqefkW1hoIee_sgivMr2 26 Precision genetic engineering using the CRISPR/Cas system ➢ What can CRISPR/Cas system do? • Gene knockout: Introduce mutations within a specific sequence within the genome o Some CRISPR/Cas systems are designed to make double stranded breaks in precise locations with a genome. o When the breaks are repaired, often mistakes (nucleotide deletions/insertions/replacements) are made, leading to Lethal Mutations within a gene. • Gene insertion: Insert a “DNA construct” in a specific location within the genome o Some CRISPR/Cas systems are designed to introduce a gene construct in a precise location. o The gene insertion occurs via homologous recombination. See Video. 27 Precision genetic engineering using the CRISPR/Cas system ➢ Components of CRISPR/Cas system / complex i) A tracrRNA (trans-activating crRNA), RNA containing a stretch of bases that provide the “stem loop” structure that binds Cas nucleases ii) A Guiding RNA (gRNA) - 20 bp nucleotide sequence (fused to tracer-RNA) - Guides the complex to the target location within the DNA (Chromosome) iii) A nuclease - A Cas protein (usually Cas9) - Makes a double stranded break in the chromosome Figure from Origene video (slide 27) 28 Workflow of one of the Origene gene editing systems: Allows inserting a gene of interest in a specific location in the genome (see next side for image) • Targeting sequence is cloned in pCas-Guide vector • Gene of interest is cloned in the donor vector which has sequences for HDR-based (Homology Directed Repair) recombination • Both vectors are co-transformed in the target organism • Gene of interest (e.g., GFP) is inserted in a specified location within the genome 29 https://www.origene.com/catalog/vectors/crispr-vectors/ge100002/pcas-guide LHA: left homologous arm RHA: right homologous arm Figure 1. Flow chart of CRISPR genome editing using HDR. CRISPR/Cas9 Genome Editing manual 30