Methods_06

Since most of MN have only one recognition site in the genome (and thus are very unlikely to cut in your gene of interest) and are very difficult to engineer, the utility of this technology is largely limited to providing a consistent site for the insertion of transgenes of interest by homologous recombination

• Zinc fingers (ZFs) are small protein structural motifs that can coordinate one or morezinc ionsto help stabilize their folds
• ZFs are involved in a wide array of biological activities including DNA and RNA binding and protein-protein interactions
• ZFs are often found in transcription factors (TF)
• Most ZF-containing TFs are dimers that recognize palindromic DNA sequences
• Most contain multiple ZFs

the motif recognizes three specific nucleotides

• Affinity purification: isolates tagged recombinant proteins on an affinity resin
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• pulldowns:isolates interactors of tagged proteins from a tissue lysate on an affinity resin preloaded with recombinant bait protein
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• co-immunoprecipitation: isolates interactors of tagged or untagged proteins using protein specific primary and secondary antibodies linked to a resin
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One binds to chromatin the other binds to DNA. Chromatin has histones and other proteins to compact it. Here, ChIP-seq can be interacting with protein or dna

DAP-seq looks at interactions between protein and naked DNA

Arbitrary... depends what you are looking for. If you have RNA and what to know what protein it binds to, then the RNA is the bait, and mystery protein is the prey.

In many cases it is trial and error to engineer a protein to recognize a particular amino acid. If you are trying to get it to recognize 6-10 amino acids, this will be even more difficulty

• isolates interactors of tagged or untagged proteins using protein specific primary and secondary antibodies linked to a resin
• 

• isolates tagged recombinant proteins on an affinity resin
• 

• isolates interactors of tagged proteins from tissue lysate on an affinity resin preloaded with recombinant bait protein
• 

• 
• 

• one-to one
• or one vs. library of proteins

• low to medium
• one protein on a resin vs. many proteins in column

low to medium

• Low*
• * one bait protein can be tested against a library of preys

• Low*
• *one bait protein can be tested against a library of preys

Fok1 nuclease

• Each ZF motif recognizes 3 specific nucleotides in target DNA
• 3+ zinc fingers often combine to assemble together
• nuclease domain (Fok1) bound to ZFs allow it to cleave ds DNA

Fok1 nuclease is a dimeric restriction enzyme that recognizes and cutsto make double strand breaks

• difficult to engineer
• limited number of natural zinc fingers
• ability to recognize longer sequences is challanging

transcription activator-like effector nucleases

highly conserved repeats derived from transcription activator-like (TAL) effectors

TAL effectors are proteins produced by plant pathogenic bacteria Xanthomonas spp and are secreted into the host to alter plant gene expression

• the amino acid sequence of each repeat region in a TALEN is largely invarient, except for two adjacent amino acids.
• These variable pair of residues recognize specific DNA base pairs
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• The amino acid sequence in the repeat region of the TAL effector has a pair of variable diresidues in an otherwise unvaried region. The pair of residues can code to bind to a specific DNA bp
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• double stranded break which can result in either:
• non-homologous end joining (NHEJ) and an indel
• homologous recombination and a knock-in of donor DNA
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• Clustered Regularly Interspaced Short Palindromic Repeats
• Cas9 is one of CRISPR-associated proteins

• Invading DNA from viruses or plasmids gets cut (by Cas proteins) into small fragments (~20bp) and incorporated into a CRISPR locus of the host genome as “spacers” amidst a series of short CRISPR repeats
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These spacers serve as memory of past invasions: the CRISPR loci are transcribed and transcripts are processed to generate small CRISPR RNAs (crRNA)

• Upon repeated infection, crRNA (hybridized with tracrRNA) guide Cas9 endonuclease to invading DNA (recognized based on sequence complementarity between the spacer and the target site), leading to viral DNA cleavage (and eventually degradation)
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Processed crRNAs consists of a variable “spacer” sequence (identical to the invader’s ”protospacer” DNA) and a fragment of the CRISPR repeat itself

It hybridizes with a second RNA, known as the transactivating CRISPR RNA (tracrRNA), and together they complex with Cas9

• The spacer-section of crRNA directs Cas9 to cleave complementary target-DNA sequences (the protospacer)
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• transactivating CRISPR RNA
• This RNA hybridized with crRNA spacer sequence and forms a complex with Cas9

• The target DNA sequence in the invading DNA is cleaved only if it is located next to the protospacer adjacent motif (PAM)
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• NGG in the Streptoccus pyogenes CRISPR system
• helps CRISPR to identify where to cut

• The only limitation is that the desired target sequence needs to precede the NGG PAM sequence (so, if there are no two GGs in your gene, you won’t be able to use conventional CRISPR/Cas9 to cut it)
• Another consideration is in potential off-target effects if your gene has a close homologue, but just a 2-nucleotide mismatch (out of 20) eliminates functionality of 95% crRNA tested

Using the 'nickase' version of Cas9 to increase the length of the sequence recognized and results in two single stranded breaks

• Have 2 CRISPR units recognize sequences around the genes you would like removed
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Cas9 nickase was fused to a reverse transcriptase

The 3’ end of gRNA was extended to include a primer (i.e., a region of homology to the sequence to be edited) and a template for DNA repair via reverse transcription -> pegRNA

Cas9 nickase was fused to a reverse transcriptase

• The 3’ end of gRNA was extended to include a primer (i.e., a region of homology to the sequence to be edited) and a template for DNA repair via reverse transcription -> pegRNA
•