Genome editing uses engineered nucleases like CRISPR-Cas9 to make precise changes to DNA. The CRISPR-Cas9 system was adapted from bacteria and archaea, who use it as part of their immune system to detect and destroy foreign DNA. It involves a guide RNA targeting a specific DNA sequence, and the Cas9 nuclease cutting both strands at that location. This double stranded break can then be repaired through non-homologous end joining or homology directed repair to edit the genome. CRISPR-Cas9 provides a simple and versatile tool for genome editing in many cell types and organisms, with applications including gene knockouts, tagging, regulation of expression, and creating disease resistance.
Genome editing uses engineered nucleases like CRISPR-Cas9 to make precise changes to DNA. The CRISPR-Cas9 system was adapted from bacteria and archaea, who use it as part of their immune system to detect and destroy foreign DNA. It involves a guide RNA targeting a specific DNA sequence, and the Cas9 nuclease cutting both strands at that location. This double stranded break can then be repaired through non-homologous end joining or homology directed repair to edit the genome. CRISPR-Cas9 provides a simple and versatile tool for genome editing in many cell types and organisms, with applications including gene knockouts, tagging, regulation of expression, and creating disease resistance.
Genome editing uses engineered nucleases like CRISPR-Cas9 to make precise changes to DNA. The CRISPR-Cas9 system was adapted from bacteria and archaea, who use it as part of their immune system to detect and destroy foreign DNA. It involves a guide RNA targeting a specific DNA sequence, and the Cas9 nuclease cutting both strands at that location. This double stranded break can then be repaired through non-homologous end joining or homology directed repair to edit the genome. CRISPR-Cas9 provides a simple and versatile tool for genome editing in many cell types and organisms, with applications including gene knockouts, tagging, regulation of expression, and creating disease resistance.
Genome editing uses engineered nucleases like CRISPR-Cas9 to make precise changes to DNA. The CRISPR-Cas9 system was adapted from bacteria and archaea, who use it as part of their immune system to detect and destroy foreign DNA. It involves a guide RNA targeting a specific DNA sequence, and the Cas9 nuclease cutting both strands at that location. This double stranded break can then be repaired through non-homologous end joining or homology directed repair to edit the genome. CRISPR-Cas9 provides a simple and versatile tool for genome editing in many cell types and organisms, with applications including gene knockouts, tagging, regulation of expression, and creating disease resistance.
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GENOME EDITING:
CRISPR Cas System
Hajira Fatima M.Sc., Biotechnology What is Genome Editing? Genome editing or Genome editing with Engineered Nucleases is a type of genetic engineering in which the DNA is inserted, replaced or remove from the genome using artificial engineered Nucleases or ‘’molecular scissors’’
The Nucleases create Specific DSB’s at desired location in the genome ,the induced breaks is repaired by natural process of homologous recombinationand non homologous end joining (NHEJ)
Why Genome Editing?
To understand the function of a gene or a protein, one interferes and monitor its effect on organism Genome Editing tools :
1. Zinc Finger Nucleases (ZFN)
2. Transcription activator like endonucleases (TALEN’s) 3. CRISPR-Cas9 History of CRISPR Cas System : CRISPR Cas System : • These are the part of Bacterial And Archea Immune System, which detects and recognise the foreign DNA and Cleave it. 1. CRISPR ( Clustered regularly interspaced short palindromic Sequence) Loci 2. Cas (CRISPR associated protein) can target and cleave DNA in a sequence specific manner • It is composed of CRISPR repeat-spacer arrays, which can be further transcribed into CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), and a set of CRISPR-associated (cas) genes which encode Cas proteins with endonuclease activity
Spacers Cas genes CRISPR repeat- spacer arrays Leader Sequence
C Protospacer Adjacent Motits (PAM) • Short Sequence of DNA (2-6bp) • 5’-NGG-3’ Mechanism in Bacteria: The CRISPR-Cas system acts in a sequence-specific manner by recognizing and cleaving foreign DNA or RNA. The defence mechanism can be divided into three stages:
(i) adaptation or spacer acquisition,
(ii) crRNA biogenesis, and (iii) target interference • Different CRISPR-cas system in bacterial adaptive immunity Type 1 (CRISPR-Cas3) and Type Class 1 3 (CRISPR-Cas10) Uses several Cas protein and crRNA
Type 2 (CRISPR-Cas9) and Type
Class 2 5 (CRISPR-Cpf1) Employ a large single component cas9 protein in conjucntion with CrRNA and TracrRNA CRISPR Cas9 Genome editing mechanism
There are two types of pathways
for repairing DSBs: NHEJ and HDR.
NHEJ pathway creates an indels,
variations by insertions and deletions of few bases during the repair of DSB’s terminal. HDR pathway uses homologous sequences of donors, either in the form of dsDNA or ssDNA, which allows more accurate gene insertion than the other. • WHAT CRISPR is capable of ? CRISPR can be used in a very large variety of cells in many different organisms, such as mammals, plants and fungi. It has been used to alter genomes in many ways, including changes in nucleotide sequences and changing the expression of genes. some of the current uses of CRISPR are
Gene Tag Anti CRISPR Knock-outs
Screening CRISPRi/a Knock-ins
What make the CRISPR the ideal genome technology?
High potency (cleavage effentiancy) and Specificity
Broad applicability to both invivo and ex-vivo applications Simple editing tool Potential one time curative tool Ability to address any site in genome for foreign genome Ability to target DNA sites simultaneously Multifunctional programmability:delete,insert,or repair gene Plant Genome Editing with CRISPR cas system First use of CRISPR/Cas systems for plant gene editing in 2013 STEPS: In plants, CRISPR/Cas9-based gene-editing consists of multiple steps • Applications of CRISPR in plants 4. Gene Wide function screening • Repression • Activation • Enhancer 1. Transgene Free Modification • Viral Encoding gene • RNP editing • Crossing/Selfing 5. Gene Expression Regulation • Transcriptional Regulation • Post Transcriptional regulation
2. Virus Resistance • Cleavage of viral genome 6. Site specific DNA Integration • Genome cleaning of Virus • Gene knock out in HR • RNP Removal • Gene knock in –NHEJ • Native gene GFP fusion • Split Cas9 3. Chromosome structure and number manipulation • Deletion • Insertion • Translocation Applications in Agriculture
1. Can be used to create high degree of genetic variability at
precise locus in the genome of the crop plants. 2. Potential tool for multiplex reverse and forward genetic study. 3. Precise Transgene integration at specific loci. 4. Developing biotic and abiotic resistance trait in crop plants. 5. Potential tool for developing virus resistance crop varieties. 6. Can be used to eradicate unwanted species like herbicide resistance weeds, insect pest. 7. Potential tool for improving polyploidy crops like potato and wheat. • References 1. *CRISPR-Ebook.pdf 2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073294/ 3. https://royalsocietypublishing.org/doi/10.1098/rstb.2015.0496 4. https://www.youtube.com/watch?v=47pkFey3CZ0 5. https://www.frontiersin.org/articles/10.3389/fpls.2020.00056/full#:~:text=G enome%20editing%20with%20CRISPR%2DCas9,plants%20that%20were %20previously%20neglected . 6. CRISPR technology in plant genome editing: Biotechnology applied to agriculture 7. https://www.mdpi.com/1422-0067/20/16/4045 Thanks you
