Genome editing techniques

This presentation will provide you insight idea about techniques of genome editing.

1. Genome Editing Techniques Vikas Verma PhD Scholar, JNKVV, Jabalpur

2. Genome editing , or  genome engineering , or  gene editing , is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations. What is Genome Editing?

3. As of 2015 four families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector -based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system .   Nine genome editors were available as of 2017. All three major classes of these enzymes—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered meganucleases—were selected by  Nature Methods  as the 2011 Method of the Year .   The CRISPR-Cas9 system was selected by  Science  as 2015 Breakthrough of the Year . Genome editiors

4. Meganucleases , discovered in the late 1980s, are enzymes in the  endonuclease  family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs ).   The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence . Meganucleases have the benefit of causing less toxicity in cells than methods such as Zinc finger nuclease (ZFN), likely because of more stringent DNA sequence recognition. One major drawback is the construction of sequence-specific enzymes for all possible sequences is costly and time consuming, as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALEN-based fusions utilize . Meganucleases

5. What is ZFN technology? Zinc fingers were first discovered in the African clawed toad ( Xenopus laevis ) in 1985 A class of engineered DNA-binding proteins Facilitate targated editing of the genome by creating double strand breaks in the DNA at specified locations Double strand breaks are importand for site-specific mutagenesis Stimulate the cell’s natural DNA repair processes i.e , HR and NHEJ Generate precisely targeted genomic editing resulting in cell lines with targated gene deletions, integrations, or modifications

6. What are zinc finger nuclease Highly specific genomic scissor Consists of two functional domains A DNA – binding domain A DNA- cleaving domain comprises of nuclease domain of FoK I

7. Diagrammatic representation of ZFN technology A pair of ZFNs, each with three zinc fingers binding to target DNA double strand break FokI domain

8. Applications of ZFN Repairing mutations Insertion of gene or DNA fragment at specific site Repair or replace aberrant genes Disabiling an allele Allele editing Applications in medical sector a) Gene therapy b)Treatment of HIV

9. TALENs :Transcription activator-like effector nucleases  TALENs are the restriction enzyme engineered to cut specific sequences of DNA They are made by fusing: DNA-binding domain (TAL effector ) DNA-cleavage domain ( the catalytic domain of RE FoK I) TALENs can be engineered to bind any desired DNA sequence to cut at specific locations in DNA

10. TALEN constructs are used in a similar way to designed zinc finger nucleases And have three advantages in targeted mutagenesis: DNA binding specificity is higher off-target effects are lower, and construction of DNA-binding domains is easier Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision .

11. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses. They consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position. Several companies, including  Cellectis and   Editas have been working to monetize the CRISPR method while developing gene-specific therapies CRISPRs

12. Components of CRISPR Proto spacer adjacent motif (PAM) CRISPR RNA ( crRNA ) Trans activating crRNA ( tracr RNA )

13.  Nuclease platforms  ZFN  TALEN  CRISPR/Cas9 Source Bacteria, Eukaryotes Eukaryotes Bacteria (Streptococcus sp.) DNA binding determinant Zinc finger protein Transcription-activator-like effector crRNA/sgRNA Binding specificity 3 Nucleotides 1 Nucleotide 1:1 Nucleotide pairing Mutation rate (%) 10 20 20 Target site length (bp) 18–36 24–40 22 Endonuclease Fok I Fok I Cas9 Double-stranded break pattern Staggered cut (4–5 nt , 5′ overhang) Staggered cut (Heterogeneous overhangs) Sp Cas9 creates blunt ends; Cpf1 creates staggered cut (5′ overhang) Off-target effects High Low Variable Ease of design Difficult Moderate Easy Dimerization required Yes Yes No Methylation sensitive Yes Yes No Best suited for Gene knockout, Transcriptional regulation Gene knockout, Transcriptional regulation Gene knockout, Transcriptional regulation, Base editing Applications Human cells, pig, mice, tobacco, nematode and zebrafish Human cells, water flea, cow and mice Human cells, wheat, rice, maize and Drosophila Comparison of ZFN, TALEN, CRISPR-Cas9 Technologies

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