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Lit review
- 1. Reviewing the applications of the CRISPR/Cas9 genome editing system in cancer resistance Presented, edited and created by Benjamin Oxley
- 2. A brief History of CRISPR • 1987- Discovery of Interspaced repeat elements by Yoshizumi Ishino The repeats can vary in size from 21 base pairs, present in Salmonella typhimurium, to 37 base pairs present in Streptococcus pyogenes.
- 3. • 2000 - This unique organization of interspaced repeat elements eventually lead to their classification as a family of Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR for short. • 2002 – Discovery of CRISPR-associated genes (Cas) To summarise, the characteristics found at the CRISPR loci by Jansen et al., (2002) were: • Multiple short direct repeats • Non‐repetitive spacer sequences between the repeats all of similar size • A common leader sequence in most species harbouring multiple CRISPR loci • The absence of long open reading frames within the locus • The presence of the cas1 gene accompanied by the cas2, cas3 or cas4 genes in CRISPR‐containing species.
- 4. • 2005 – It was hypothesized that CRISPR was an adaptive immune system. This hypothesis was proved in 2007. • 2005 – Cas 9 discovery There are three major types of CRISPR systems and they can be easily classified due to their exclusive proteins: type 1 = Cas 3; type 2 = Cas 9 and type 3 = Cas 10.
- 5. Type 2 CRISPR/Cas system mechanism of action 1. Firstly Cas9 associates with guide RNA (gRNA) 2. The gRNA will direct the Cas 9 to a Protospacer Adjacent Motif in the invading virus/plasmid which it will bind 3. Once bound to PAM the gRNA will unwind part of the double helix allowing Cas 9 to cut it. 4. Once Cas 9 has cut the DNA and produced the double stranded breaks (DSBs), the cell will try to fix the damage via repair mechanisms.
- 6. NHEJ
- 7. HDR
- 8. Genome editing • Much interest began to grow around the potential of applying the type 2 CRISPR system into genome editing after it was shown to be transferable into different bacterial strains. Gasiunas et al., 2012, demonstrated that they could reprogram Cas9 to target a site of their choosing by changing the sequence of the crRNA and in 2013 (Zhang et al.,) demonstrated that the system could be programmed to target multiple genomic loci.
- 9. Applying the type 2 system Overcoming drug resistance in cancer: Cancerous cells can acquire Antineoplastic resistance to anti-cancer therapies. 1. KBM5 is a myeloid leukemia cell line that develops resistance to imatininb 2. This cell line develops resistance because it doesn’t express ASXL1 due to a point mutation. 3. This resistance can be overcome by cloning single guide RNA’s (sgRNAs) into a vector that encodes for Cas 9 and transfecting the vector into KBM5 cells. 4. The results of this transfection were a high degree of CRISPR/Cas9-based mutation correction
- 10. Another example Trastuzumab is another antibody used in cancer treatment. It targets human epidermal growth factor 2 (HER2) in breast cancers but unfortunately after a while resistance builds up. To solve this resistance a better understanding of it’s mechanism was required. 1. CRISPR-Cas9 was used to knock out ARID1A in BT474 breast cancer cells 2. ARID1A knockout lead to the expression of Annexin A1 (ANXA1) 3. High expression of ANXA1 then activates AKT in the HER2/PI3K/mTOR pathway, which finally produces drug resistance. The results of this work showed that ANXA1 high tumors are less responsive to HER2/PI3K/mTOR–targeting strategies and may benefit from including inhibitors targeting AKT in their treatment regimen.
- 11. Careful considerations and possible problems • Length of sgRNA’s • Type of sgRNA • Choice of delivery method • Off-target effects
- 12. The future I’m aspiring for a career in cancer research as I personally know the horrible effects it has on families and communities and with the rapid rise in the use of the type 2 CRISPR system hopefully I will one day be fluent with it’s use in some kind of genome editing type role.
- 13. Thank you for listening.
Editor's Notes
- Whilst attempting to clone the iap gene, Ishino came across a set of 29 nucleotide repeats, which can be seen by the hairpin like structures. The repeats caught the attention of Ishino due to their uncharacteristic grouping as they were interspaced by 5 intervening, 32 nucleotide long nonrepetitive sequences instead of the typical arrangement of repeat sequences where they are consecutive to the DNA.
- Using a set of bioinformatic tools Jansen ad co attempted to compare the CRISPR loci in different bacterial, archaeic, eukaryotic and viral genomes. CRISPR were only found to be present in the bacterial and archaic strains but were more prevalent in the bacteria. This lead to identifying AT rich leader sequences, several hundred base pairs long located at one side of the CRISPR loci. They also found four genes which were shown to flank the CRISPR loci and termed them Ca1 - 4. Cas 1 was found in all genomes containing a CRISPR loci and it showed to have characteristics of DNA binding proteins although its precise function was unknown NO ORF = DON’T CODE FOR PROTEINS.
- This was shown by analysing the CRISPR sequences of different bacterial strains for variances in the type and number of spacers and Upon infection with virulent bacteriophages The CRISPR became resistant by integrating spacers derived from the phage DNA 2005 also marked the discovery of a new Cas gene that could encode a large protein predicted to have nuclease activity. This was known as Cas 9
- Which is a complex of crRNA and tracrRNA. The crRNA will contain a spacer region which serves to differentiate between the invading virus/plasmid and the bacterial DNA because the viral genome will contain a protospacer which corresponds to the spacer. 2. This binding makes sure that it only cleaves the PAM and not the spacer sequence within the CRISPR array in the bacterial DNA. 3. Cutting is carried out by Cas 9’s RuvC and HNH domains which cleave the non-target and target DNA strands respectively 4.
- This process begins with resection which is when small regions degrade away from either side of the double stranded break. This is followed by the recruitment of several proteins that bind to the broken ends, leaving single base pairs from the break exposed Artemis is then recruited trims away the single stranded tail overhangs and then ligase IV joins the broken ends together.
- Homologus recombination In the same way as NHEJ this begins with resection only around the 5’ end by the MRN-CtIP-complex leaving a 3’overhang. The 3’ overhang is then coated with RPA before being replaced by Rad51 Rad51 mediates strand invasion on a homologous template forming a holliday junction. The double Holliday junctions are then converted into recombination products by nicking endonucleases,
- Due to ease of its ease of use and low cost, the type 2 crispr/cas system has been applied in many genome editing experiments. Including reducing drug resistance in cancer. Can build up after exposure to treatment or may e down to certain genetic traits. 1) Imatininb is a tyrosine kinase inhibitor given to sufferers of myelocytic leukemia. 2) ASXL1 is a tumour suppressor involved in regulating transcription of genes involved in proliferation 3) This lowers resistance because the sgRNA’S were designed to target a region that overlapped the ASXL1 mutation in the KBM5 cells. 4)Leading to high rates of ASXL1 expression in the KBM5 cells and lowered imatininb resistance.
- ARID1A knockout cell lines were generated using a dual vector doxycycline-inducible CRISPR/Cas9 vector system and gRNA targeting ARID1A ARID1A is a member of the SWI/SNF family, and is thought to regulate the transcription of certain genes. Mutations that inactivate it have been found in a lot of cancers ANXA1, a member of the annexin calcium/phospholipid-binding protein family which is involved in cell proliferation and tumor progression
- sgRNAs with more than 85 nucleotide long tracrRNA tails could result in an increased amount of indels . truncated guide RNA should be also avoided as it reduces Cas9’s cutting activity. A common method is The introduction of plasmids that simultaneously encode sgRNA and Cas9 into target cells by electroporation but this can lead to all or part of the plasmid integrating into the host genome. Which could effect function. CRISPR/Cas9 system can produce a substantial amount of off‐target mutagenesis which impacts precise gene modification