genetic engineering CRISPR gene knockdown (nisha).pptx

CRISPR gene knockdown

1. Nisha Kalra 7 th semester Department of Microbiology Faculty of Life Sciences (FLSI) BUITEMS Gene Knockdown

2. Introduction An adaptive immune system in bacteria, have been modified for genome engineering Prior to CRISPR, genome engineering approaches like zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs) required scientists to design and generate a new nuclease pair for every genomic target Comparative simplicity and adaptability of CRISPER

3. Cont … Mainly two components: a guide RNA (gRNA or sgRNA) a CRISPR-associated endonuclease (Cas protein) The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ∼20 nucleotide spacer that defines the genomic target to be modified Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA.

4. Generating a Knockout Using CRISPR By co-expressing an endonuclease like Cas9 a gRNA specific to the targeted gene The genomic target can be any ∼20 nucleotide DNA sequence, provided it meets two conditions: The sequence is unique compared to the rest of the genome. The target is present immediately adjacent to a P rotospacer A djacent M otif (PAM) The PAM sequence serves as a binding signal for Cas9, but the exact sequence depends on which Cas protein you use

5. Cont … Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9 Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.

6. Cont … Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal to the target DNA Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains, called RuvC and HNH, to cleave opposite strands of the target DNA The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence)

7. Cont … The resulting DSB is then repaired by one of two general repair pathways: The efficient but error-prone non-homologous end joining (NHEJ) pathway The less efficient but high-fidelity homology directed repair (HDR) pathway

8. The NHEJ repair pathway The most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations. In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene.

9. Making Precise Modifications Using Homology Directed Repair (HDR) While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.

10. Cont … A DNA repair template containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) Depending on the application, the repair template may be a Single-stranded oligonucleotide, Double-stranded oligonucleotide, or A double-stranded DNA plasmid

11. Cont … The efficiency of HDR is generally low For this reason, many laboratories try to enhance HDR by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ may also increase HDR frequency. Since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of the Cas9-induced DSBs will be repaired via NHEJ

12. Applications Knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line MDR in cancer is generally the result of increased expression of membrane efflux proteins which belong to the ATP-binding cassette (ABC) transporters superfamily Consequently, cancer cells actively transfer a wide variety of pharmaceutical compounds, including chemotherapeutic agents out of the cells, resulting in reduced intracellular drug accumulation in resistant cells Classical multidrug resistance to chemotherapeutic agents is attributed to the overexpression of ABCB1 gene (known as P-glycoprotein (P- gp ) or MDR1) It has been documented that overexpression of P- gp is the key factor for reduced chemo-sensitivity in a wide range of tumors, including ovarian cancer Desired genetic modifications could be achieved by introducing a double-strand break (DSB) at a specific target sequence followed by stimulation of cellular DNA repair pathways, including error-prone non-homologous end joining (NHEJ) and the homologous recombination (HR)

13. Improving muscle function in mice with Duchenne muscular dystrophy with CRISPR Researchers have used CRISPR genome-editing tools to treat muscular dystrophy in mice They removed the defective gene in mice with Duchenne muscular dystrophy using CRISPR; through this, they allowed the animals to produce one of the major proteins in the muscles However, the use of CRISPR seemed impossible in mice that had the disease already, because mature muscle cells generally do not divide and, as a result, they do not necessarily have the DNA repair machinery to add or correct DNA However, CRISPR can remove the wrong exon so that the duplicator machine can produce a shorter version of dystrophin functioning like its normal version sgRNA and Cas9 were transferred by an adenovirus carrier into rat muscle cells; then the wrong exon was removed using the CRISPR system

14. Resistance against malaria by modification of mosquito DNA using the CRISPR technique Using CRISPR, researchers have developed mosquitoes that transmit resistance to disease in the species of their own Using DNA editing, scientists injected engineered CRISPR to Anopheles mosquitoes, which are one of the major malaria-carriers in Asia The inserted DNA encoded engineered antibodies that attack the malaria parasites. In the laboratory, this feature was extended to 99.5 percent of the offspring from mating between modified and unmodified mosquitoes

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