Gene Editing 1 PDF

Gene Editing  DNA loops D-loop T-loop  Genome Editing Targeted interventions at the molecular level of DNA, deliberately to alter the structural or functional characteristics of organisms Site-Directed Nucleases (SDN): ✓ Zinc Fing...

Gene Editing  DNA loops D-loop T-loop  Genome Editing Targeted interventions at the molecular level of DNA, deliberately to alter the structural or functional characteristics of organisms Site-Directed Nucleases (SDN): ✓ Zinc Finger ✓ TALENs (transcription activator-like effector nucleases) ✓ CRISPR/Cas9 systems (Clustered Regularly Interspaced Short Palindromic Repeats - associated protein-9 nuclease (Cas9)) ✓… Zinc Finger Nucleases as Potential Reagents to Create Double- Strand Breaks in Normal Genes FokI nuclease domain (Fn) FokI nuclease domain (Fn) Initially developed by labs of Srinivasan Chandrasegaran (Johns Hopkins) and Dana Carroll (Univ. Utah) Structure of TAL Effector & TALEN RVD’s Introduction to CRISPR http://www.addgene.org/CRISPR/guide/ (Bhattacharya et al. 2023)  Introduction to CRISPR short palindromic sequences 10  Introduction to CRISPR Spacer Clustered Regularly-Interspaced Short Palindromic Repeats CRISPR 11  3 distinct types of bacterial CRISPR systems identified so far: – Type I, II, III – Type II is the basis for current genome engineering applications From Streptococcus pyogenes The S. pyogenes system is orthogonal to the native E. coli system Jinek et al., Science 2012  Identification of cas genes Addison V. Wright, et al. 2016, Cell Cas, CRISPR associated proteins CRISPR confers adaptive immunity (Jiang et al. 2016) (Bhattacharya et al. 2023)  Genomic manipulation requires only : – the Cas9 protein and – an engineered small guide RNA (sgRNA) with a PAM sequence upstream of target complementary sequence Contains a designed hairpin to mimic the CRISPR RNA and trans-acting RNA complex Base-pairing between the sgRNA and target DNA causes double- strand breaks due to the endonuclease activity of Cas9 http://www.addgene.org/CRISPR/guide/ Qi et al. Cell 2013  DNA target does not need to be unique and can appear in multiple locations, all of which will be targeted by the Cas9 nuclease for cleavage – can be on a plasmid or integrated into the bacterial genome. The sgRNA can bind on either strand of DNA and the Cas9 will cleave both strands (double strand break, DSB). The DSB results in the silencing of that DNA sequence. Changing the target specificity of the RNA-protein complex does not require protein engineering but only the design of the short crRNA guide Directing the specificity of dual-RNA:Cas9 with several different crRNAs allows for the introduction of multiple mutations at the same time. Jiang et al. Nature Biotech, 2013 Cpf1, a Single RNA-Guided Endonuclease Cpf1, CRISPR from Prevotella and Francisella 1 Cpf1 makes staggered cuts Cpf1 cuts at a distal site T-rich PAM Cpf1 may be easier to deliver to cells Zetsche B, et al., Cell, Oct. 2015, 163(3):759-771 Fagerlund R D, et al., Genome Biology, 2015, 16(1):1-3 Prime Editor Type of CRISPR action Genome engineering Transcriptional regulation RNA targeting Others  Type of CRISPR action Cut Base Edit Nick dCas9-FokI  Type of CRISPR action Activate  Type of CRISPR action Interfere Epigenetics  Type of CRISPR action RNA Targeting RNA Editing  Type of CRISPR action Purify Genomic Loci Visualize Genomic Loci Type of CRISPR action  Gene Editing with CRISPR/Cas9 NHEJ: non-homologous end joining HDR: homology directed repair Wiles et al., Mamm Genome (2015)  Conventional Gene Targeting in ES Cells WT GeneX NeoR DTA Spontaneous homologous recombination Targeted NeoR with marker Cre or FLIP Targeted w/o marker ES cell targeting CRISPR in zygotes Application requires an ES cell line Any strain Editing frequency very rare targeted gene editing Drug selection yes no Locus dependence variable low Time to heterozygotes 8-12 months ~6 months Scarless editing no yes Conventional Gene Targeting vs. CRISPR/Cas Gene Editing - Targeting - Synthesis of construct guide RNA, Cas9 & donor - ES cells construct electroporatio n & selection - Colony picking & expansion - Screening - Blastocyst - Injection into injection 1-cell embryos - Embryo transfer - Embryo transfer Chimera Mosaic Founder Revised from Liu et al., EMBO reports (2017)