GILESTRO - zfn/talen/crispr Flashcards
Gene targeting by HR is improved by ZFN, TALEN, & CRISPR
Induce ds breaks in the DNA to trigger cellular repair machinery
o Can result in:
§ Indel mutations of the gene by NHEJ
§ Introduction of new DNA sequences/ precise gene editing if the cell is also provided with a modified template (Homology-directed Repair)
* More efficient homologous recombination
Introduction of ds breaks by engineered endonucleases
Zinc Finger Nucleases (ZFN)
TALEN (transcription activator-like effector nuclease)
CRISPR/Cas9
Zinc Finger Nucleases (ZFN)
- Discovered from observing TF-DNA interaction, which bind DNA in a specific way, involving a Zinc Finger domain that fits the curvature of DNA
- Each Zinc finger domain bind to 3 nucleotides in a DNA sequence – if not all of the 3 interactions are right, the ZF won’t bind well.
- Multiple ZF domains are fused with nucleases to cut DNA at the specific sequence the ZFs recognize
- ZFN are designed to act as a pair for specificity (ensure that there are no off-site double cuts – each ZFN can cause off-site single cuts which will not be damaging to DNA)
o Therefore, need to design & create 2 ZFN that recognize the region flanking both left and right of the target cleavage site (difficult)
To create ZFN:
- Select a protein with ZF domain known to have some affinity to target DNA sequence
- With the help of computer programming, modify the ZF to generate a first product with some binding efficiency to the target DNA sequence
- Perform in vitro evolution: introduce the ZF into bacteria and make it such that the bacteria will only survive if the introduced ZF binds the target sequence in bacterial DNA (ex. make the ZF bind upstream of an antibiotic resistant gene and grow the bacteria in the antibiotic, so only bacteria with ZF efficient in binding DNA will survive)
- This selective environment promotes evolution and mutation in the ZF as the bacteria grows to improve binding and ensure bacterial survival, resulting in a ZF with efficient binding property
o Problem: great binding property of the ZF results in off-site binding as well, so another round of in vitro evolution can be performed to select for ZF that specifically binds the target sequence - Fuse the acquired ZF to a nuclease, producing 1 ZFN that nicks ssDNA (repeat the process for another ZFN that nicks ssDNA in the other flanking region of target sequence)
TALEN (transcription activator-like effector nuclease)
- Use TAL effectors derived from bacteria that infect plants ex. rice & bell peppers
- Also works in pairs to ensure specificity but simpler to make
- TAL effectors contain repetitive units that form the DNA binding domain. Each subunit contact 1 nt of DNA and does not interfere with each other’s binding. Each subunit is identical except at 2 amino acids. Variation in those result in different affinity to the nucleotides
TALEN can be created by:
- Order TAL units known to bind the specific nucleotides in order of the target DNA sequence – results in TAL effector that binds the target sequence
- Fuse the TAL effector with a nuclease to form TALEN
Size of the recognition region is restricted to 15 bases to mimic the natural shape and size of the TAL effector protein
CRISPR/Cas9
Best technique cus always use the same protein (cas9) despite diff. target sequences
* Utilize guide RNA complementary to the target sequence to guide the protein to the target sequence of cleavage
2 components of CRISPR/Cas9
- Cas9 - DNA cutting protein
* has 2 active sites that recognize the guide RNA - Guide RNA
* Has 2 regions: 1 that recognizes Cas9 and 1 that is complementary to the target DNA sequence
* Easy to obtain by oligoPCR
Form a complex so guide RNA guides Cas9 to target DNA sequence where Cas9 can perform its DNA cutting activity at the target site. Cas9 introduces ds breaks by default due to presence of 2 nuclease domains.
Cas9 can be engineered to
- Have 1 nuclease domain instead to only introduce ss break (become nickase)
* Increase specificity so 2 Cas9 are needed to recognize 2 flanking regions of the sequence
*off-site ss break = able to repair and won’t cause as much affect as off-site ds break
* Error w/ ds break can only occur if the 2 nickase have off-sites close enough to each other - happens when there is similar repetition of the desired sequence in the genome
ex. members in the same gene family - Replace one of its nuclease domains with a specific enzyme
* the enzyme is guided to perform its activity on the target sequence
* Ex. deaminase – can mutate specific DNA bases (ex. replacing C with T) used as specific gene editing for repair of disease-causing point mutations to healthy version of gene OR to introduce STOP codon at specific place - Regulate transcription
* By deactivating Cas9 completely so it can no longer cut DNA, but transcriptional activators/ repressors are added to Cas9 (either directly or via a string of peptides or by guide RNA)
* These activators/ repressors can then recruit cell transcription machinery to the desired sequence to regulate transcription - Have Attached GFP
* To tag specific sequences with GFP to identify the sequence location in the chromosome – can be used to visualize 3D architecture of genome, to paint the chromosome, and to follow the chromosome’s position in nucleus
Limits of target sequence:
- need to start with GC pairing (stronger than AT) and need to end with a PAM sequence of CC for Cas9 recognition
- Can use other family of other Cas to not be restricted by PAM sequence – each Cas has diff PAM requirements
CRISPR induced ds break can result in:
- NHEJ – error prone ds break repair system – result in indels
- HDR – introduce an exogenous template that contains a modified region of interest for cell to use for repair – the region has flanking homologous ends so it can undergo HR to be integrated into the genome – result in precise genome editing
CRISPR = very efficient
– can directly perform somatic CRISPR that affect a specific cell type– is not required to be performed in germ line cells and does not require selection of offspring
* Ex. can utilize GAL4/UAS system
o Use fly line with GAL4 that is only expressed in the brain
o Fuse with fly line with UAS that promotes cas9 expression
o Gets Cas9 cuts in the brain
CRISPR/Cas9 is a natural process in bacteria
- It is the bacterial adaptive immune defense for protection against virus and phages
- Has Cas genes, tracrRNA, & CRISPR locus
o Cas gene codes for the Cas proteins
o CRISPR locus codes for pre-crRNA transcript – contain multiple CRISPR sequence complementary to specific viral sequences (spacers) interspersed between nonvariable sequences (short palindromic repeats)
o tracrRNA bind to Cas9 protein & is also complementary to the non-variable
region of CRISPR locus
Involves tracrRNA:crDNA co-maturation & Cas co-complex formation
- tracrRNA guides Cas9 protein to each non-variable region between the different CRISPR sequences, which along with RNase3, will cleave the pre-crRNA transcrip (contain many spacers between short palindromic repeats) into smaller mature crRNA transcripts (contain 1 spacer, 1 palindromic repeat)
- Each newly cleaved crRNA transcript will remain bound to the tracrRNA as Cas9 forms complex with other Cas proteins
- Each crRNA guides the Cas9 & Cas complex to a specific invading viral sequence (protospacer) downstream of PAM for Cas9 to cleave the sequence into fragments
- If the cleaved viral fragment slightly differs from the viral sequence in crDNA used to recognize it, the new sequence can also be inserted into the CRISPR locus by the Cas protein. Thus, from this day on, the bacteria has acquired knowledge to defend against this other viral sequence.
in biotechnology CRISPR,
the guide RNA is artificially created to mimic the crRNA:tracrRNA pairing (1 molecule that already has 2 regions to both bind Cas – tracrRNA function & recognize target sequence - crRNA function)
Different types of CRISPR
Type 1: require a complex of multiple proteins. The cascade complex, a large multi subunit complex, binds to target DNA. Inside the complex, usually is a small cas6 subunit that processes pre-crRNA. Also includes a protein (nuclease) that performs DNA cleavage.
Type 2 (Cas9 system):
Requires RNase3 that processes precursor RNA. It cleaves long pre-crRNA into smaller mature crRNAs. Involves cas9 protein which is guided by tracrRNA to cleave & process pre-crRNA. crRNA then guides Cas9 to perform ds cleavage on target sequence.
Type 3
* Cas6 processes pre-crRNA & transfer crRNA to CMR which binds & cleaves target DNA
