NucleicAcids19 CRISPR Cas9 Genome editing.pdf

BIOC 3041 Nucleic Acids Biochemistry CRISPR/Cas9 – A breakthrough in gene editing technology Lecture Recap • RNAs can serve as regulators of gene transcription and translation • sRNAs can serve to repress levels of one protein and promote another • Bacterial mRNA translation can be regulated by s...

BIOC 3041 Nucleic Acids Biochemistry CRISPR/Cas9 – A breakthrough in gene editing technology Lecture Recap • RNAs can serve as regulators of gene transcription and translation • sRNAs can serve to repress levels of one protein and promote another • Bacterial mRNA translation can be regulated by sRNA binding to key regions • A gene’s transcripts can directly sense metabolite concentration changes • Activated riboswitch structures block further transcription or any translation • Riboswitches can bind and thus respond to simple and complex metabolites C.R.I.S.P.R.s are a Record of Infections Survived, of Resistance Gained Clustered Regularly Interspaced Short Palindromic Repeated DNA sequences Where are CRISPRS found? • Genome sequencing finds them in ~50% of bacteria, 90% of Archaeons How are CRISPRs structured? 1) ~500 bp ‘leader sequence’ (includes a promoter) 2) ~30 bp highly conserved repeats 3) ~30 bp highly divergent sequences • Number of CRISPR clusters ranges from 1 per genome (usually) but up to 20 in more unusual cases, but up to 400 in extreme example CRISPR regions are structured, recognizable DNA sequences in non-eukaryotes Where do CRISPR Spacer DNA Sequences Originate From? • Divergent spacer sequences’ source was unknown • Bioinformatics revealed spacer sequence DNA was from bacteriophages (viruses that target bacteria) and plasmids • CRISPR system now known to be defence system against foreign (viral) DNA How to prove this? 1. Bacteria infected with virus gain new DNA from infecting viruses 2. Can add in new viral DNA to make “naïve” bacteria virus-resistant 3. Can delete existing spacer (viral) DNA, bacteria are now virus-susceptible CRISPR DNA sequences are key part of adaptive immunity in non-eukaryotes CRISPR sequences partnered with accessory proteins CRISPR-Associated (Cas) proteins required for defence against foreign DNA • Cas and other proteins are part of set, but not all are present in all genomes • These are suite of proteins needed for defense against, acquisition, addition, and use of foreign DNA • Cas1 “integrase” and Cas2 exonuclease present in all systems, needed for defence, acquisition • Other Cas, Cmr, Cse, Csx proteins used, but not essential in all cases CRISPR accessory Cas proteins required for integration of foreign DNA Spacer Sequences are acquired from Infecting Viruses • Invading foreign DNA is processed by Cas2 nuclease, part of which becomes new protospacer between two repeat sequences • Protospacer-Adjacent Motif (PAM) is part of Repeat • Cas1 integrase will integrate this new protospacer into host DNA • Other Cas proteins involved in expression, processing of existing spacers or antiviral defence • Can delete Cas-1 & -2 genes but host can only defend itself against viruses from which viral DNA already acquired (can’t learn new tricks) Foreign DNA is captured and turned against itself by integrating it into genome CRISPR transcribed as long RNA subject to processing for targeting invading DNA or RNA • Leader sequence contains promoter for driving CRISPR transcription which contains spacers, repeats • CasE processes the pre-crispr RNA (pre-crRNA) • Resulting short crRNAs=length of 1 spacer + 1 repeat • crRNAs have 5′ and 3′ “handles”, made of the repeats • RNA “handles” (aka tracRNA) serve as binding sites for proteins that form the Cascade complex targeting the foreign DNA • “guide” RNA binds complementary strand in target DNA sequence (spacer ensures specificity) • Cas9 nuclease cleaves both of DNA helix’s 2 strands CRISPR RNA modified, used by Cas proteins to target DNA CRISPR is mimicked in animals by Piwi-Associated RNAs • Bacteria, Archae use the CRISPR system to target hostile foreign DNA • Animals use Piwi-associated RNAs (piRNAs) to target transposons to suppress them • Transposon remnants are 45% of our genome, kept suppressed by histone modification and then formation of heterochromatin • Transposons (“jumping genes”) targeted by piRNAs presumably made from transposons • Failure to destroy transposons leads to spontaneous mutagenesis Animals defend against detrimental RNA using CRISPR-like piRNAs and Piwis 2020 Nobel Prize in Chemistry Jennifer Doudna Emmanuelle Charpentier • www.youtube.com/watch?v=avM1Yg5oEu0 • Joins many other Nobel Prizes in Chemistry that focus on nucleic acids biochemistry (DNA double helix, DNA & RNA polymerases, DNA sequencing, restriction endonucleases, catalytic RNA, DNA repair, ribosomes….) • Stunning video 1st to show CRISPR editing DNA in real time using atomic force microscopy https://www.youtube.com/watch?v=gvTbIdKFJMM CRISPR/Cas9 are exploited to generate different mutations • Guide RNA (crRNA) matches targeted DNA and allows Cas9 to cut both strands • Double-strand breaks are made and prompts DNA damage response • Recall highly mutagenic NHEJ • If donor DNA is present or supplied, new DNA can be inserted into site • Allow scientists/doctors to break/fix genes CRISPR/Cas9 now used to create ‘knock-out’ or ‘knock-in’ mutations Overview of how CRISPR and Cas proteins are used Virus DNA 1. Acquisition leader 1 2 3 Plasmid DNA 4 CRISPR array n Cas Cas locus 2. Expression 3. Interference Pre-crRNA Cas proteins crRNA www.youtube.com/watch?v=2pp17E4E-O8 1:07 onwards Cas9 mutant proteins exploited to repress/activate target genes • Cas9 protein can be modified • Cas9 can be disabled so DNA is bound using guide RNA but not cut • Allows delivery of easily-designed custom DNA-binding protein complex • Transcription Repressor/Activator proteins fused to Cas9 alter gene expression in controlled fashion Cas9 mutant proteins can be improved to increase specificity • Cas9n ‘nickase’ mutant cuts only once • Requiring 2 different Cas9/guide RNAs means each complex needs to be close for gene editing • Prevents problem of off-target cuts! www.youtube.com/watch?v=4YKFw2KZA5o Mutated Cas9 nucleases increase possibilities of genetic engineering Newest CRISPR technique, Prime Editing CRISPR • Prime editing reduces “off-target” hits, doesn’t used DSB repair or need to supply donor DNA with desired edits (published 2019, ~3000 citations!) • Catalytically broken Cas9 is fused to reverse transcriptase • Prime guide RNA encodes targeting sequence which encodes desired edits Reverse transcriptase • Original opposite complementary strand nicked, identified for “repair” by removal and re-synthesis to match new engineered strand • New method can fix insertions/deletions, all 12 types of mutations • “In principle, (method) could correct 89% of known pathogenic genetic variants” New CRISPR Prime Editing greatly improves prospects for genetic surgery CRISPR and genetic alteration of humans • Nucleosomes can limit gRNA-Cas9 access to PAM sequences in nucleosome-wrapped DNA • CRISPR/Cas9 nucleases can induce many ‘offtarget’ mutations, but efficient gRNA design and follow-up testing can reduce them • First alteration of humans done in 2018 without approval, may have unknown side effects… • Sickle-cell anemia, hypercholesterolemia treatments in human fixed by CRISPR, no offtarget effects detected – yet…. Permanent human genetic alteration now a reality 2019 Therapy increases production of fetal haemoglobin, which can reverse sickle-cell disease and β-thalassaemia by isolating a patient's stem cells from blood and then editing the cells with CRISPR-Cas9 designed to disrupt BCL11A, a regulatory protein that switches cells from fetal to adult hemoglobin And as the 1 yr anniversary of her landmark treatment approaches, Gray has received good news: The billions of genetically modified cells her body appearofto “Talking about the future is better than letting itdoctors sneakinfused up on into us. We needclearly to do more be alleviating virtually all thepositive complications this or we will be left with very limited vocabulary in the space between and of heron disorder, sickle cell negative hype.” - George Church, speaking the potential ofdisease. CRISPR technology Where might this go? www.youtube.com/watch?v=PC6ZA1dFkVk Recap of lecture • CRISPR regions are structured, recognizable DNA sequences in non-eukaryotes • CRISPR DNA sequences are key part of adaptive immunity in non-eukaryotes • CRISPR accessory Cas proteins required for integration of foreign DNA • Foreign DNA is captured and turned against itself by integrating it into genome • Animals defend against detrimental RNA using CRISPR-like piRNAs and Piwis • CRISPR RNA modified, used by Cas proteins to target DNA • CRISPR/Cas9 now used to create knock-out or knock-in mutations • Mutated Cas9 nucleases increase possibilities of genetic engineering • Permanent human genetic alteration now a reality • New CRISPR Prime Editing greatly improves prospects for genetic surgery Reading – MBOG7 pg706-711, 721-725 MBOG6 doesn’t cover this FYI – will not be tested • Podcast: An anti-CRISPR system • Some viruses have found a clever way to inhibit bacteria’s CRISPR–Cas immune system. They use small pieces of RNA that mimic part of the system to stop it from attacking the virus’s genetic material. The discovery could have applications in phage therapy, in which bacteria-infecting viruses are used in place of antibiotics. “If we are able to equip phages with anti-CRISPR strategies, they would be more effective at taking over bacterial populations and killing nasty bugs,” microbiologist and study co-author Rafael Pinilla-Redondo tells the Nature Podcast. • https://www.nature.com/articles/s41586-023-06612-5 • 00:47 An RNA-based viral system that mimics bacterial immune defences • To protect themselves against viral infection, bacteria often use CRISPR-Cas systems to identify and destroy an invading virus’s genetic material. But viruses aren’t helpless and can deploy countermeasures, known as anti-CRISPRs, to neutralise host defences. This week, a team describe a new kind of anti-CRISPR system, based on RNA, which protects viruses by mimicking part of the CRISPRCas system. The researchers hope that this discovery could have future biotechnology applications, including making CRISPR-Cas genome editing more precise.