CRISPR/gene editing Flashcards
Jansen et al 2002
- In 2002, a group of Dutch researchers (Jansen et al.) found that the genomes of bacteria and archaea contain clusters of repetitive palindromic elements that are interspaced by short non-repetitive sequences. They named these elements Clustered Regularly Interspaced Short Palindromic Repeats or CRISPRs. These CRISPRs were flanked by genes of unknown function that contained helicase and nuclease domains, which they called Cas (or CRISPR-associated genes)
Jinek et al 2012
Showed that CRISPR Cas9 system can be used to precisely cut defined dsDNA sequences in a test tube
Three independent groups 2005
found that the spaces in CRISPR loci were bacteriophage-derived. This was picked up scientists at the Danish yoghurt company, Danisco, and this was the first scientific evidence that the CRISPR/Cas system actually functions as a prokaryotic adaptive immune system
Swiech et al. (2014)
delivered AAV expressing Cas9 and gRNA targeting MeCP2 into the hippocampal DG of adult male mice. Using next-generation sequencing, they found indel mutations in 68% of targeted cells. Inactivation of MeCP2 significantly impaired contextual memory (contextual fear conditioning paradigm), which indicates a role for MeCP2 in DG neurons in contextual memory.
Deglon et al
have been studying the potential therapeutic value of CRISPR/Cas9 genome editing in a Huntington’s disease mouse model of expressing mutant huntingtin; they used viral vectors to infect neurons with Cas9 and gRNA targeting huntingtin. Initial indications seem positive; CRISPR treatment reduced brain Htt aggregates by 90%. However, both mutant and normal alleles are targeted, so efforts are currently underway to selectively target mutant alleles.
The team has recently developed a promising alternative to CRISPR called KamiCas9. It uses two guide RNAs, one to target the huntingtin gene and the other to target the Cas9 gene. Thus Cas9 can be permanently be inactivated after it has targeted the mutant huntingtin gene. This reduced the off-target effects when applied to neural cells from HD patients
Chen et al 2013
showed in a landmark study that it is possible to dynamically visualise genomic loci in living cells using a CRISPR system that uses dCas9 fused to eGFP. Interestingly, they showed that binding specificity of Cas9 dramatically increased by making small modifications in the constant region of the guide RNA.
- This technique could be highly useful for visualising the 3D architecture of the genome, or to paint an entire chromosome and follow its position in the nucleus
Qin et al. (2017)
used a single dCas9 and a modular set of sgRNAs to visualise chromatin loci throughout the cell cycle and determine the different positions of transcriptionally active and inactive regions in the nucleus.
Ma et al. (2015)
used differently coloured dCas9-sgRNAs in live human cells to image chromosomal loci in multicolour. Using this method, they could determine the intranuclear distance between loci on separate chromosomes, and could assess DNA compaction in different regions of the cell
Liu et al 2018
fused dCas9 to Tet1 (demethylating enzyme) to reverse the hypermethylation associated with FMR1 silencing in FXS-derived iPSCs.
With CCG-sgRNA, the dCas9-Tet1 complex was targeted to the CGG expansion, which induced demethylation of the upstream FMR1 promoter
Chromatin from heterochromatin → active state
increase in FMR1 expression to 90% of wild-type levels, and FMRP was upregulated to 73% of wild-type levels
When engrafted into mouse brain, FMR1 expression was maintained in these edited neurons at 3 months
Han et al 2020
in which they performed CRISPR screens in 2D monolayers and 3D lung-cancer spheroid models in order to reveal cancer vulnerabilities.
o Their analyses revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. They also identified that carboxypeptidase D expression was correlated with outcomes of lung cancer patients, and that decreased carboxypeptidase D expression reduced tumour growth.
Chen et al. (2018)
developed Cas12a into a diagnostic tool named DETECTR that can detect specific DNA sequences in patient samples.
o They showed Cas12a could target a specific locus within the human papillomavirus (HPV) genome by using small fluorescently-quenched single-stranded DNA reporters that produce a fluorescent signal when Cas12a pairs with its DNA target
o They also demonstrated that DETECTR could accurately distinguish between HPV-16 and HPV-18 from human cells at attomolar levels within one hour
Broughton et al. (16th April 2020)
published in Nature a paper explaining how they have used the DETECTR system to detect SARS-CoV-2 from respiratory swab RNA extracts
o They included 36 patients with COVID-19 infection in their study and 42 patients with other viral respiratory syndromes as controls
o Their DETECTR assay provided a faster (takes less than 40 minutes) and visual alternative to the US CDC real-time RT-PCR assay
o Their method had 95% positive predictive agreement and 100% negative predictive agreement
Garriga-Canut et al 2012
In one of the first studies, preceding CRSIPR/Cas9 tech development, this group used ZFPs to selectively bind and repress expanded CAG repears in the R6/2 mouse model of HD
Lombardo et al. (2007)
published the first successful study using ZFNs and donor sequences to correct a point mutation in haematopoietic stem cells in the IL2RG gene which causes leukaemia in X-linked severe combined immunodeficiency (SCID)
2017, Brian Madeux
Had Hunter’s syndorme - recieved ZFN with an AAV for in vivo genetic editing - first example of this in humans
