<p>Synthetic
biology reprograms organisms to perform non-native functions for beneficial
reasons. An important practice in
synthetic biology is the ability to edit DNA to change a base pair, disrupt a
gene, or insert a new DNA sequence. DNA
edits are commonly made with the help of homologous recombination, which
inserts new DNA flanked by sequences homologous to the target region. To
increase homologous recombination efficiency, a double stranded break is needed
in the middle of the target sequence.
Common methods to induce double stranded breaks use nucleases, enzymes
that cleave ribonucleotides (DNA and RNA).
The most common nucleases are restriction enzymes, which recognize a
short, fixed, palindromic DNA sequence (4-8 base pairs). Because of the short and fixed nature of the
recognition sites, restriction enzymes do not make good candidates to edit
large chromosomal DNA. Alternatively,
scientists have turned to programmable endonucleases which recognize user-defined
DNA sequences, often times much larger than the recognition sites of
restriction enzymes (15-25 base pairs).
Programmable endonucleases such as CRISPR-based systems and prokaryotic
Argonautes are found throughout the prokaryotic kingdom and may differ
significantly in activity and specificity. To compare activity levels among
endonuclease enzymes, activity assays are needed. These assays must clearly delineate dynamic
activity levels of different endonucleases and work with a wide variety of
enzymes. Ideally, the activity assay
will also function as a positive selection screen, allowing modifications to
the enzymes via directed evolution. Here, we develop an <i>in vivo</i> assay for programmable endonuclease activity that can also serve
as a positive selection screen using two plasmids, a lethal plasmid to cause
cell death and a rescue plasmid to rescue cell growth. The lethal plasmid houses the homing
endonuclease, I-SceI, which causes a deadly double-stranded break at an 18 base
pair sequence inserted into an engineered <i>E.
coli</i> genome. The rescue plasmid
encodes for a chosen endonuclease designed to target and cleave the lethal
plasmid, thereby preventing cell death.
With this, cell growth is directly linked to programmable endonuclease
activity. Three endonucleases were
tested, SpCas9, eSpCas9, and xCas9, displaying recovered growth of 49.3%,
26.1%, and 16.4% respectively. These
values translate to kinetic enzymatic activity and are congruent with current
literature findings as reported values find WT-SpCas9 to have the fastest
kinetics cleaving around 95% of substrate within 15 seconds, followed closely
by eSpCas9 cleaving 75% of substrate within 15 seconds and finally trailed by
xCas9 cleaving 20% of substrate in about 30 seconds. The differences between
each endonuclease’s activity is exacerbated in our <i>in vivo</i> system when compared to similar <i>in vitro</i> methods with much lower resolution. Therefore, slight differences in activity
between endonucleases within the first few minutes in an <i>in vitro</i> assay may be a few percentages different whereas in our <i>in vivo</i> assay, these differences in
activity result in a more amplified signal. With the ability to display the dynamic
response of enzymes, this assay can be used to compare activity levels between
endonucleases, give insight into their kinetics, and serve as a positive
selection screen for use in directed evolution applications. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/11328623 |
| Date | 06 December 2019 |
| Creators | Michael A Mechikoff (8088809) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/DEVELOPMENT_OF_AN_ASSAY_TO_IDENTIFY_AND_QUANTIFY_ENDONUCLEASE_ACTIVITY/11328623 |
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