L22 CRISPR Handouts PDF

CRISPR-Cas gene editing tool https://www.criver.com/products-services/discovery-services/screening-and-profiling-assays/assay-development/crispr-gene-editing?region=3701 Therapy for sickle cell anemia as the illustrativ...

CRISPR-Cas gene editing tool https://www.criver.com/products-services/discovery-services/screening-and-profiling-assays/assay-development/crispr-gene-editing?region=3701 Therapy for sickle cell anemia as the illustrative example § Sickle cell anemia – which gene to “edit” for treatment § CRISPR-Cas gene editing system – an introduction § Why do bacteria have CRISPR-Cas systems? § Using CRISPR-Cas for gene editing Int. J. Mol. Sci. (2021) 22:3327  Casgevy and Lyfgenia, both are cell-based therapies Lyfgenia: HbA-T87Q gene introduced through lentivirus vector. This protein binds O2 just as HbA does + has anti-sickling properties because of the T87Q mutation https://www.lyfgenia.com/what-is-lyfgenia/how-lyfgenia-works https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510747/ https://www.theweek.in/health/more/2023/02/25/sickle-cell-anaemia-causes-history-prevention-treatment-in-india.html § Severe pain, organ damage, and shortened life span § Anaemia → end-organ damage and premature death. § Painful blood vessel blockages (vaso-occlusive crises, VOCs) Results in severe and debilitating pain Leads to acute chest syndrome, stroke, jaundice, heart failure symptoms https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-announce-authorization-first § Largely symptomatic treatments focussed on relieving pain minimizing organ damage maintaining hydration addressing fevers § Requires medication and sometimes monthly blood transfusions § Cure: a stem cell transplant from a matched donor Available only to a small fraction of people https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-announce-authorization-first  Secondary Primary and Tertiary Quaternary Function Structure Structures Structure 1 Normal 𝛃 Normal Proteins do not associate 5 µm subunit hemoglobin with one another; each 2 carries oxygen. 3 𝛃 Normal 4 𝛂 5 6 7 𝛃 𝛂 Figure 5.19. Campbell Biology. 10th ed. By Reece and others  Secondary Primary and Tertiary Quaternary Function Structure Structures Structure Sickle-cell 𝛃 Sickle-cell Proteins aggregate into a 1 5 µm subunit hemoglobin fiber; capacity to 2 carry oxygen 3 𝛃 Sickle-cell is reduced. 4 5 𝛂 6 7 𝛃 𝛂 Figure 5.19. Campbell Biology. 10th ed. By Reece and others  Depend upon the number of mutant alleles one has inherited Sickle cell alleles Sickled RBC Low O2 Sickle cell disease Sickle cell Fiber formed hemoglobin by sickle cell hemoglobins Figure 14.UN08. Campbell Biology. 10th ed. By Reece and others § Sickle cell anemia is caused by a single mutation in the gene encoding b-subunit of Hb § Adult hemoglobin – HbA – subunit composition is a2b2 § Fetal hemoglobin – HbF – subunit composition is a2g2 Fetal hemoglobin: HbF (a2g2) Adult hemoglobin: HbA (a2b2) 1. a or z 2. b, g, d or e https://onlinelibrary.wiley.com/doi/full/10.1110/ps.072891007 Given § Level of fetal hemoglobin (HbF) is a well characterized ameliorating factor § BCL11A, a transcription factor, is the principal regulator of the development switch from HbF to HbA Idea (or plan for the treatment) § Can resetting the “BCL11A switch” lead to HbF production instead of HbA? Turn off BCL11A expression: g subunit is expressed, b subunit is not BCL: B-cell lymphoma https://www.pnas.org/doi/10.1073/pnas.0711566105 https://ashpublications.org/blood/article/128/19/2338/35512/ Factors that control the expression of BCL11A 1 2 3 BCL11A Proteins + factors that work with BCL11A Switch off g subunit expression Switch on b subunit expression Downstream pathways https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870126/  Effect at the level of RBCs HbF production increases The process of producing RBCs (erythropoiesis) is normal https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870126/  Effect at the level of an organism (mouse model) Prevents and reverses g subunit production Corrects and prevents sickle cell disease phenotypes No abnormalities in the production of RBCs https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870126/  Effect at the level of an organism Patients with “genetic deletions” containing the BCL11A locus (three such individuals) BCL11A haploinsufficiency increases HbF Individuals with deletions in the region where BCL11A gene is present Normal hematologic and immunologic parameters Neurodevelopment role identified Deletion region: 2p15-p16.1 Patient 1: ∼440 kb deletion Patient 2: ∼1 Mb deletion Patient 3: ∼875 kb deletion BCL11A gene is deleted in all three Haploinsufficiency: situation wherein one copy of a gene is inactivated or deleted and the second https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497765/ functional copy is not adequate to produce the needed gene product to preserve normal function. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870126/ § Sickle cell anemia is quite prevalent, especially in India § No cure as of now... Only management provided diagnosed § FDA of USA has approved gene therapy for treatment § BCL11A – a transcription factor encoding gene – is the target § There are different classes, types, and sub-types of CRISPR-Cas systems § In this lecture, we focus on CRISPR-Cas9, the most studied system § CRISPR and Cas are acronyms Acronyms are abbreviations that may be pronounced as words Clustered CRISPR Regularly Interspaced Associated Short System Palindromic Repeats AIIMS vs IIT UNESCO vs WHO  5’-GAATTC-3’ Level 3’-CTTAAG-5’ Palindromes because Malayalam 5’-GGATCC-3’ sequences (in 5’ to 3’ Madam, I’m Adam 3’-CCTAGG-5’ direction) are the same in both the strands Able was I ere I saw Elba 5’-GGCGCGCC-3’ 3’-CCGCGCGG-5’ Not a palindrome (*) 5’-GACCAG-3’ Sequence in the 1st strand: 5’-GACCAG-3’ 3’-CTGGTC-5’ Sequence in the 2nd strand: 5’-CTGGTC-3’ ere means “before” in (old) English (*) Referred to as a mirror repeat or mirror-like palindrome Elba = Island where Napoleon Bonaparte was exiled  Interspaced repeats Tandem repeats 5ʹ 3ʹ R R R R R R 3ʹ 5ʹ The same sequence repeats again and again 5ʹ 3ʹ 3ʹ 5ʹ A specific A bacterial genome R sequence of DNA  Interspaced repeats 5ʹ 3ʹ R R R R R 3ʹ 5ʹ § A palindrome § Length: 20 to 40 bp (numbers from different sources vary slightly) § Sequence similarity: high among related species § Overall, highly diverse sequences  Interspaced repeats 5ʹ 3ʹ R S R S R S R S R 3ʹ 5ʹ § Spacers (denoted by the letter S) § Length: 20 to 60 bp (numbers from different sources vary slightly) § Each spacer is from a “foreign organism”  Context: CRISPR 5’------CGGTTTATCCCCGCTGCGCGGGGAACTC------3’ 3’------GCCAAATAGGGGCGACGCGCCCCTTGAG------5’ 1. Strand separation 2. Transcription 5’------CGGTTTATCCCCGCTGCGCGGGGAACTC------3’ Coding strand Transcribed RNA 5’------CGGUUUAUCCCCGCU.... Template strand 3’------GCCAAATAGGGGCGACGCGCCCCTTGAG------5’  Context: CRISPR 5’------CGGUUUAUCCCCGCUGCGCGGGGAACUC------3’ Transcribed RNA G C § A stem-loop structure U C G § 3D structural motif Dot = WC H-bonding G C C G Recognition + e.g., C G binding by Cas C G C G C G U A 5’------CGGUUUA ACUC------3’ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847661/ https://www.biorender.com/icon/short-hairpin-rna-shrna-2  CRISPR: Cluster of Regularly Interspaced Short Palindromic Repeats Cluster of (i) Regularly Interspaced, (ii) Short, (iii) Palindromic Repeats This region of the genome has a § Cluster of repeats + few other ”elements” § Repeats are 1. Regularly Interspaced by spacers (spacers are DNA from foreign organisms) 2. Short (20 to 40 bp) 3. Palindromic Electron micrograph (partial) (sequence reads the same in both strands) of a bacterial genome https://commons.wikimedia.org/wiki/File:DNA_Under_electron_microscope_Image_3576B-PH.jpg  Cas genes (3) AT-rich leader sequence Required for transcription of CRISPR 5ʹ 3ʹ 3ʹ 5ʹ tracrRNA Palindromic repeats + spacers 5ʹ 3ʹ R S R S R S R S R 3ʹ 5ʹ The number of Cas genes and the location + direction of transcription of tracrRNA (vis-à-vis other elements of the locus) vary in different classes, types, and sub-types https://crisprcas.i2bc.paris-saclay.fr/Home/About § A CRISPR locus consists of the following: 1. Cas genes (number varies from 3 to 13+) Many classes, types, and sub-types based on Cas gene variants 2. Leader sequence AT-rich region Promoter for transcription of CRISPR i.e., spacer + palindrome 3. CRISPRs (spacers + palindromes) 4. Trans-acting CRISPR RNA (abbreviated as tracrRNA)  A CRISPR § Each CRISPR is unique in terms of Palindromic repeat (length, sequence) Spacers (host pathogens) Cas (number of genes, mode of action, etc.) tracrRNA (sequence, relative location and Electron micrograph (partial) direction of transcription) of a bacterial genome Location (in a genome or a mega plasmid) https://commons.wikimedia.org/wiki/File:DNA_Under_electron_microscope_Image_3576B-PH.jpg  Embryonic Adult stem stem cells cells Totipotent Multipotent Cultured stem cells Different culture conditions Liver cells Nerve cells Blood cells Stem cells are also found in adult brain, skin, hair, eyes, and dental pulp, but in smaller amounts. (Campbell, p426) Figure 20.20. Campbell Biology. 10th ed. By Reece and others, p426 Isolate HSPCs Hematopoietic Stem and Progenitor Cells Front. Pediatr. (2023) 11:1249275 Ex vivo gene therapy Delete BCL11A gene using CRISPR-Cas Administer by micro-injection mRNA for Cas9 1. tracrRNA 2. BCL11A gene fragment as spacer 3. Palindromic repeat #1, #2, and #3 are fused together as “single guide RNA” (sgRNA) Front. Pediatr. (2023) 11:1249275 Ex vivo gene therapy Deleting BCL11A using CRISPR is but one of the methods One can use lentiviruses also to introduce edited gene (Prof. Rahul Purwar’s CAR-T cell therapy uses this method) It is also possible that the mutant b- subunit gene is edited, instead of deleting BCL11A gene Front. Pediatr. (2023) 11:1249275 Condition the recipient (patient) 2-weeks prior to infusion Chemotherapy, radiation, etc. “Cleaning up” the bone marrow Enable gene-edited cell to proliferate Front. Pediatr. (2023) 11:1249275 Infuse gene-edited HSPCs Front. Pediatr. (2023) 11:1249275 Int. J. Mol. Sci. (2021) 22:3327 Genome editing Machine learning High-throughput Live cell data generation imaging Science (2023) 379: eadd8643 (2023)  30 Dec 2019 Sentencing... https://www.science.org/content/article/chinese-scientist-who- produced-genetically-altered-babies-sentenced-3-years-jail 31 July 2024 Optimism... 26 Nov 2018 Birth of gene edited twins https://www.science.org/content/article/crispr-bombshell- chinese-researcher-claims-have-created-gene-edited-twins https://www.technologyreview.com/2024/07/ 31/1095509/he-jiankui-hopeful-gene-editing/