Question

Use the spacUse the space below to compare and contrast genome editing by Crispr‐Cas9, ZFNs, and TALENs. What are the similarities, differences, advantages, and disadvantages of each technologye below to compare and contrast genome editing by Crispr‐Cas9, ZFNs, and TALENs.  What are the similarities, differences, advantages, and disadvantages of each technology

Answer 1

CRISPR/Cas9

CRISPR/Cas9 CRISPR/Cas9 system is a bacterial defence mechanism against bacteriophage infection. When a viral dna( Bacteriophage , in this case) integrates into the bacterial genome, it produces RNA which is taken up by Cas9.

Advantages

high speed,zero limit, high efficiency, high quality, diversification, no species limitation

Disadvantage
One of the challenges of using CRISPR-Cas9 editing technique is offtarget effect where Cas9 enzymes cut wrong genes. Targeting specificity of Cas9 is known to be tightly controlled by twenty nucleotide guided sequence of the SgRNA and the presence of protospacer adjacent motif (PAM) next to the target sequence in the gene, potential off target cleavage activity could still occur (50% chance) on DNA sequence with even 3-5 base pair mismatches.

Zinc-finger nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside Cas9 and TALEN proteins, ZFN is becoming a prominent tool in the field of genome editing.

Advantages

• Rapid disruption of, or integration into, any genomic loci
• Mutations made are permanent and heritable
• Works in a variety of mammalian somatic cell types
• Edits induced through a single transfection experiment
• Knockout or knock-in cell lines in as little as two months
• Single or biallelic edits occur in 1–20% of clone population
• No antibiotic selection required for screening

Transcription activator- like effector nuclease

Transcription activator-like effector nucleases (TALEN)ctivator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations.[1] The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

TALENs – Transcription activator-like effector nucleases - are similar to ZNFs in that they use DNA binding motifs to direct the same non-specific nuclease to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide. The interactions between the TALEN-derived DNA binding domains and their target nucleotides are less complex than those between ZNFs and their target trinucleotides, and designing TALENs is generally more straightforward than ZNFs.

A key advantage of both techniques is that they are not limited to mutagenesis in mouse embryonic stem cells. ZNF and TALEN modifications have been engineered in zebrafish, fruit flies, nematodes, rats, livestock and even in monarch butterflies.