crispr Flashcards
discovery of CRISPR studies (6)
Charpentier and Doudna awarded Nobel Prize in Chemistry 2020 for discovery
Key paper = Jinek et al (2012) showed, using surveyor nuclease assays, that CRISPR/Cas9 system (from Strep. thermophilus or pyogenes) can be used to cut user-defined DNA sequences (CLTA gene) in vitro highlighted potential for gene editing
- Ground-breaking = previous methods (Res) only recognise 1 small DNA sequence, usually 6bp long, which occur thousands of times within a given genome CRISPR can be programmed to recognise and cut much longer DNA pieces = enabling cutting of 1 unique sequence within genome
Cong et al (2013) and Mali et al (2013) = generated CRISPR/Cas9 system allowing genome editing in mammalian cells in vivo (human or murine cells)
Wang et al (2013) = use CRISPR/Cas9 system to generate mouse models (see below)
Later studies used CRISPR/Cas9 to create mutations in living mice (not inducing germline mutations but correcting specific mutant genes in adult mice)
- E.g. Long et al (2014) = correcting non-sense mutation in Dmd gene causing Duchenne’s muscular dystrophy, Yin et al (2014) = correcting of LOF mutation in FAH associated with hereditary tyrosinemia
first animal models made using CRISPR
Wang et al (2013)
• used CRISPR/Cas9 to generate mouse models carrying germline biallelic mutations in 5 loci (Tet 1, 2, 3, Sry, Uty)
• Took as little as 3 weeks (normally takes between 6 and 12 months), with efficiency of up to 80%
• Transcribed components into mRNA and directly injected into zygotes implanted into foster mothers
CRISPR/Cas9 to make cancer models
Platt et al (2014) = showed CRISPR/Cas9 could be used to rapidly model dynamics of multiple mutations in tumorigenesis
• Created Cre-dependent Cas9 mouse = overcome challenges associated with Cas9 delivery
- Inserted Cas9 transgene expression into Rosa26 locus = interrupted by loxP-STOP cassette so inducible by Cre recombinase, linked to enhanced GFP (allow visualisation of Cas9-expressing cells)
- Designed AAV vector containing sgRNAs targeting 3 most commonly mutated genes in human lung adenocarcinoma = induced mutations in KRAS, and KO of p53 and LKB1
- Delivered intratracheally = detected 1.3% p53 indels, 1.3% LKB1 indels, 1.8% KRAS mutations
- Two months post-injection, 100% of mice developed spectrum of lung nodules (vs 0% of mice with control AAV)
example of CRISPR library screen
• E.g. Hart et al (2015)
- Tested Toronto knockout library (composed of 176500 sgRNAs against 17661 protein-coding genes (approximately 10 sgRNAs/gene) in 5 human cell lines
- Deduced that 829 genes are essential for cell fitness
- Useful = helps inform design of future KO cell lines (determined whether particular gene KOs will be lethal or not)
CRISPR/Cas9 library screen for cancer
• Lau et al (2020)
- Treated HAP1 cell line with spectrum of anti-cancer drugs, mutagenized cells with human CRISPR Knockout v2 library
- Selected resistant cells using minimal lethal concentration = sgRNA abundance was quantified relative to unselected control population = identified significantly enriched hits
- Importantly identified 10 multi-drug resistance genes (e.g. MSH2, BAX), including uncharacterised gene named Required for Drug-induced Death 1 (RDD1) = loss resulted in resistance to 5 drugs
- Targeting RDD1 led to chemotherapy resistance in mice = increased tumour growth and shortened lifespan
- Also found that significant reductions in RDD1 expression were observed in multiple cancers (e.g. breast, colorectal, lung, ovarian) = associated with patient outcome
dCas9 and telomeres
• Chen et al (2013) = used dCas9 fused to GFP to dynamically visualise specific DNA sequences on telomeres
- Labelling efficiency and intensity similar to that achieved by FISH = current gold-standard
- In vivo CRISPR imaging allowed us to track particular DNA sequences within cell = study of telomere dynamics during elongation + disruption, subnuclear localisation of MUC4 loci, cohesion of MUC4 loci on sister chromatids, dynamics during mitosis
dCas9 and FXS
An example of the potential use of dCas9 fused to chromatin modifiers is for the potential treatment of fragile X syndrome (FXS),
• FXS is the most common inherited cause of mental retardation, caused by a CGG repeat expansion in the fragile X mental retardation (FMR1) gene, which triggers epigenetic silencing of the gene by hypermethylation loss of expression of the Fragile X mental retardation protein (FMRP) usually expressed widely, particularly in neurons
• No treatment or cure for FXS exists, but a new approach is to try and reactivate FMR1 expression using CRISPR tools, by removing the epigenetic marks that sustain FMR1 transcriptional silencing
• Liu et al. (2018) – fused dCas9 to Tet1 (demethylating enzyme) to reverse the hypermethylation associated with FMR1 silencing in FXS-derived iPSCs.
o With CCG-sgRNA, the dCas9-Tet1 complex was targeted to the CGG expansion, which induced demethylation of the upstream FMR1 promoter
o This switched the repressive heterochromatin status of the FMR1 promoter to an active chromatin state increase in FMR1 expression to 90% of wild-type levels, and FMRP was upregulated to 73% of wild-type levels
o Neurons derived from these methylation-edited FXS iPSCs had a restored wild-type phenotype and multi-electrode array assays revealed electrophysiological abnormalities were rescued
o When engrafted into mouse brain, FMR1 expression was maintained in these edited neurons at 3 months
o Moreover, demethylation of CGG repeats in post-mitotic FXS neurons also reactivated FMR1
• This study shows that demethylation of the CGG using dCas9 technology is sufficient for FMR1 reactivation, and targeted epigenome-editing in this manner has potential as a therapeutic strategy for FXS.
• However, the studies so far have all been in cell models – there are important considerations for clinical application
o Unknown safety
CGG repeats are common in the genome could produce off-target effects
Also, whilst over 200 CGG repeats in the FMR1 gene FXS via a loss-of-function mechanism, permutation alleles consisting of 55-200 repeats cause overproduction of FMR1 mRNA but a reduction of FMRP, which is associated with increased risk of Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) and Fragile X-associated Primary Ovarian Insufficiency (FXPOI)
o Delivery into the brain remains a challenge – e.g. concerns surrounding use of viral vectors
o Therapeutic window – epigenetic silencing of FMR1 occurs at approximately 11 weeks gestation, so it is unclear whether FMR1 silencing can be reversed in cells in which the FMR1 gene has already been switched off
- So still significant obstacles to overcome
ex vivo editing
2019 = CRISPR therapeutics
• Conducted first successful clinical trials targeting somatic cells using CRISPR/Cas9 based therapy
• Cured disease in 2 patients with beta-thalassemia and SCA = both caused by mutations in B-globin gene, resulting in RBCs inefficient at carrying oxygen = require lifelong transfusions and associated chelation therapy to remove extra iron
• Targeted BCL11A gene in stem cells of patient
- BCL11A = transcription factor that inhibits production of foetal haemoglobin
- Inducing deletion in BCL11A using Cas9 production of foetal haemoglobin no longer repressed sickling of red blood cells prevented
• Then performed autologous HSC transplant of corrected cells
• First patient treated is now transfusion-free after 9 months of treatment, with sustained HbF >10g/dL (previously required 17 blood transfusions a year)
• Highlight extraordinary potential of CRISPR to become possible permanent solution in restoring disease-causing genetic defects = solves underlying cause rather than just alleviating symptoms temporarily
gene editing in vivo
Congenital blindness trial (in vivo) (2022) = BRILLIANCE run by Editas
• Removal of point mutation (IVS26) in CEP290 gene = causative gene for type 10 leber congenital amaurosis (LCA) = rare + incurable retinal degenerative disease, causing defects in retinal photoreceptors and severe vision loss
• Uses adenovirus vector AAV5 to deliver CRISPR components under promoter specific to photoreceptors, administered via subretinal injection
• September 2021 = Editas revealed adult patients had not experienced serious adverse events or dose-limiting toxicities, modest signs of visual improvement in some patients in mid-dose group, side effects such as hypotony, retinal tears and retinal haemorrhage in low and mid-dose groups
• Critique of method
- AAV vectors can randomly integrate into genome
- unclear how patients’ immune system will respond to bacterially derived CRISPR-Cas0 components
- concern about off-target effects
- will manipulations be stable long-term?
CXCR4/CC45 editing (ZFNs)
e.g. CXCR4/CCR5 editing to facilitate HIV immunity
Didigu et al (2013)
• Used zinc-finger nucleases to genetically modify CCR5 and CXCR4 in primary human CD4+ T cells induced dose-dependent reduction in surface expression of both coreceptors
• Cells proliferated normally and were highly resistant to infection by both R5 and X4-using HIV in vitro
• ZFN disruption also provided protection against HIV in humanised mouse model
- NSG immunodeficient mice infused with WT CD4 T cells, or CD4 T cells treated with R5-ZFN or both ZFNs then infused with CD4 T cells infected with R5 and X4 viruses
- ZFNs facilitated better control of CD4+ T cell counts = CD4 counts in mice from R5/X4-ZFN group were 200-fold higher than in other groups 55 days post infection
• Translation to humans?
• Problems with ZFNs = prone to off-target cleavage so extensive screening required to ensure optimal editing of target genes without toxicity = expensive
CXCR4/CCR5 (CC9)
Yu et al. 2018
• used CRISPR/CAS9 to knock-out CCR5 and CXCR4 in human primary CD4 T cells = disrupted >12% of CCR5 and 10% of CXCR4, conferred resistance to both -X4 and -R5 HIV strains, no discernible mutagenesis in top-ranked off-target genes by Surveyor nuclease analysis and NGS
• important as it has been shown that CXCR4 trophic HIV strains are present in chronically infecting HIV-1 individuals, so both needed for functional cure
• CD4 cells chosen over CD34 (HSC) cells because of challenges gene editing stem cells whilst retaining pluripotency and concerns over purity and functionality seen in gene edited CD34 IPSCs. CD4+ cells seen as easier to edit and cultivate in vitro as well as stem-cell like subsets, central memory cells effector memory cells and stem-cell like memory cells enabling self-renewal of this population of cells.
CAR T cells
Eyqyem (2017) = used CRISPR/Cas9 to generate TRAC CAR T cells
- Problems with current methods = randomly integrating vectors can lead to oncogenic transformation, variegated gene expression and transcriptional silencing, expression of endogenous TCR can lead to autoimmunity and alloreactivity
- Here = treated T cells with Cas9 mRNA and gRNA against TCR alpha constant (TRAC) locus via electroporation = 70% KO efficiency with limited cell death = then transduced with AAV vector containing self-cleaving P2A peptide and anti-CD19 CAR cDNA for CAR knockin
- 95% of CAR+ cells were TCR-, no off-target hotspots revealed by NGS
- Better in murine model of B cell precursor ALL treated mice with TRAC CAR T cells, RV-CAR T cells in which TRAC locus was disrupted, and RV-CAR T cells in which TCR locus was not disrupted after euthanasia at day 17, higher numbers of CAR T cells, better tumour control, fewer markers of exhaustion
- Statistically significant prolonged median survival at all doses
- Using CRISPR/Cas9 to target CAR to TCR locus could provide safer, more potent therapeutic
PD-1 CRISPR edited T cells
Lu (2020)
- Used CRISPR/Cas9 to KO PD-1 (targeting exon 2) in patients’ T cells in 12 patients with treatment resistant NSCLC
- 36.3% reduction in PD-1 expression (FC), NGS = very few off-target effects,
- Safe and feasible = no grade ¾ adverse effects, maintained in patients for 52 weeks, median overall survival = 42.6 weeks
- But no objective response = perhaps due to lack of antigen specificity, tumour independence from PD-L1, too few in number
Cas12a
• Chen et al (2018)
- Can use Cas12a as diagnostic tool = named DETECTR to detect specific DNA sequences in patient samples
- When Cas12a cuts target DNA, it spontaneously cuts ssDNA in close proximity (trans-cleavage)
- In a proof-of-concept experiment, they targeted Cas12a with a crRNA to a specific DNA sequence within the human papilloma virus (HPV) genome
- At the same time, they added a small fluorescently quenched ssDNA reporter, similar to what is used in Taqman qPCR arrays = produces a signal when the ssDNA is degraded
- To enhance sensitivity, the DNA is first amplified through RPA, which is an isothermal alternative to PCR
- They observed that when Cas12a-cRNA base pairs with its dsDNA target, the non-specific ssDNA activity of Cas12a is initiated which cleaves the quencher, resulting in a quantifiable fluorescent signal and indicates the presence of the DNA of interest, in this case HPV
- Rapid and accurate test to detect carcinoma-associated HPV types 16 and 19 from clinical specimens
cas13a
• Gootenberg et al (2017) = can use Cas13a as diagnostic tool too named SHERLOCK = allows for detection of 3 ssRNA targets and one dsDNA target in single reaction
- Accurately detected Zika ssRNA, synthetic ssRNA, DENV ssRNA and synthetic dsDNA in less than 90 minutes
