MIC115 Recombinant DNA Cloning Lecture 14 Notes PDF

MIC115 Recombinant DNA Cloning 24FQ Lecture 14 – Genome editing Homologous recombination and non-homologous end joining Text with gray bars: you do not need to remember SUGGESTED VIDEOS Gen...

MIC115 Recombinant DNA Cloning 24FQ Lecture 14 – Genome editing Homologous recombination and non-homologous end joining Text with gray bars: you do not need to remember SUGGESTED VIDEOS Gene Editing Mechanism of CRISPR-Cas9 https://wyss.harvard.edu/media-post/gene-editing-mechanism-of-crispr-cas9/ Non-homologous end joining https://www.youtube.com/watch?v=31stiofJjYw LECTURE 14 TOPICS Two major DSB repair pathways (HR vs. NHEJ) Genome editing by non-homologous end joining (NHEJ) Genome editing by homologous recombination (HR) LECTURE 14 LEARNING GOALS Understand two major DSBs repair mechanisms: HR vs. NHEJ Describe how NHEJ leads to small deletions Understand the outline of HR-directed genome editing Explain how CRISPR-Cas9 technology facilitates genome editing I. DNA double-strand breaks (DSBs) repair DSBs are one of the most deleterious DNA lesions in cells Two major pathways for DSBs repair: HR (homologous recombination): error-free, template (homology)-based, using sister chromatids Predominant repair in germ cells to maintain genomic integrity. NHEJ (Non-homologous end joining): Error-prone: random repair (not based on homology), Predominant repair in somatic cells II. NHEJ (Non-homologous end joining) “Unligatable” ends occur after DSBs. Therefore, trimming or filling is needed to allow ligation. NHEJ repairs DSBs but can make mutations (primarily deleted sequences due to trimming). NHEJ contributes to mutagenesis. (do not need to memorize detailed pathways) III. HR (homologous recombination): HR in mitotically dividing cells: Using sister-chromatids (identical; generated after DNA replication): 1 MIC115 Recombinant DNA Cloning 24FQ Error-free, template (homology)-based, Maintenance of genome integrity. (do not need to memorize detailed pathways) HR in mitotically dividing cells requires a homologous template (sister chromatids)– in S phase or G2. In S phase, there are early and late replicating regions, so more regions can support HR as S phase progresses HR in meiotic cells: Using homologous chromosomes (One from mother and the other from father are very similar in sequence, but not identical): Contributes to the genetic diversity Crossovers (between homologous chromosomes) shuffle maternal and paternal alleles on a specific chromosome in meiosis (do not need to memorize detailed pathways) HR can take place between similar sequences (At least 1000 bp long: 95% homology) located in different chromosomes. HR can cause ectopic crossing over between similar sequences. This aspect explains why gene manipulation can be possible by HR. IV: Genome editing: DSBs repair is the trigger HR using Exogenous DNA: Genomic DNA can be modified based on exogenous DNA. HR can be used gene targeting, knock-in, large deletion, knockout (i.e., complete loss-of- function of target genes). Trimming is necessary for NHEJ: Thereby, NHEJ creates small deletions. Small deletions often cause frameshift mutation and complete loss-of-function of target genes (knockout). The CRISPR-Cas9 technology (used from 2013) can induce DSBs at a specific site. CRISPR- Cas9 provides a means to make gene targeting much more efficient by generating DSB(s) at a specific site. V: CRISPR genome editing The Nobel Prize in Chemistry 2020, Emmanuelle Charpentier and Jennifer A. Doudna CRISPR–Cas9 can mediate sequence-specific DSBs Simplified CRISPR/Cas9 nuclease system: 1. The Cas9 nuclease (bacterial protein): Nuclease 2. Single guide RNA: Short RNA (~20 nucleotides) These two components can induce sequence-specific DSBs We can induce site-specific DSB repair 2 MIC115 Recombinant DNA Cloning 24FQ One rule: PAM (protospacer adjacent motifs) : 5’-NGG-3’ = AGG, CGG, GGG, or TGG Must be present at 3’end of templates CRISPR-Cas9 mediated DSBs can be repaired by HR or NHEJ. VI: Genome editing by HR HR can be used for targeted insertion of DNA molecules into model organisms - yeast - mammalian cells HR can be used to target an exogenous DNA molecule to a gene in the genome and cause that gene to be altered. Usually, flanking sequences of homology (at least 20 bp long is necessary). HR is utilized to make knockout and knock-in in mammalian cells A potential approach to gene therapy in humans. Mammalian cells: HR works best on longer substrates, and this reinforces specificity Budding yeast S. cerevisiae: modifying the genome is particularly easy because recombinant DNA with 20-nt flanking sequences can be efficiently exchanged for the homologous chromosomal site in the recipient cell. 1. Transform yeast cells with the disruption construct: homologous recombination between the ends of the construct and the corresponding chromosomal sequences replaces the target sequence with the marker gene. 2. The resulting heterozygous diploid yeast cells can be identified by their drug resistance (such as using G418). 3. Sporulation of the diploid yeast cell generates four haploid spores, with two of the spores carry only the disrupted allele. 3