University Name BBT 570 Pre-Lab Report: CRISPR Gene Modification

This pre-lab report details the application of CRISPR technology for gene editing, focusing on the modification of the ctrl gene. It covers the design of guide RNA (gRNA), the fundamental principles of gene disruption, and the experimental procedures involved. The report outlines the design process of gRNA, including identifying genomic regions of interest and selecting protospacers to minimize off-target effects. It then describes the lab procedures, including the use of PCR for nucleotide synthesis, purification, gel electrophoresis, and methods for measuring concentration and calculating molar concentration. The report also discusses optical density assessment and yeast transformation techniques. Safety considerations and references to relevant research are also included. This report provides a comprehensive overview of the CRISPR-Cas9 system and its application in gene editing.

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BBT 570 Pre-Lab Report
I. Background
Modification of Class 2 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems has
enabled the formation of guide RNA. The CRISPR is an adaptive bacterial formulated from genome
engineering. The gRNA comprises of short synthetic RNA which has scaffold sequences key for Cas
binding and defined nucleotide spacers. This has made it possible for changes to occur in the Cas protein
by making changes to the target sequence in the present gRNA (Fu et al., 2014).
II. Design of gRNA
The fundamental goal of gRNA is the disruption of gene editing purposes. This is made possible to
distinguish form effective and ineffective gRNA which have a greater ability to enhance target zone. The
initial step in designing gRNA entails identifies the genomic region of interests. Finding present axons is
key to this process. In this case, our target gene is ctrl. The next step is to find protospacers sequences.
The next step is the selection of the least protospacers which are geared towards minimizing off-targets
to increase the rate of gene knockout creation (Doench et al., 2016).
III. Lab Description
a.Overview
Employing CRISPR technology is crucial for engineering the DNA edits on the ctrl gene. Employing the
hammer gene knock out by NHEJ is essential in this protocol. Gene knocks out with CRISPR is
accomplished on the Cas9 dsDNA breaks after a cut, on the error-prone nature of the homologous gene
towards the end-joining of the NHEJ, disrupting the coding of the DNA gene. The target gene will be the
ctrl gene which is responsible for fo the neurosporene synthesis from the phytoene and the lycopene.
Cutting on this gene repression on the gRNA is essential to reduce the carotene production process.
The sequence of the nucleotide used was designed based on the codon used in the gRNA without any
modification on the amino acids. The ctrl gene can encode 492 amino acids which are divided into
several sections. Each part was synthesized. The terminus was essential in designing and coming up to
complement each other (Ran et al., 2013).

b.Safety
Despite many renowned benefits towards human health, CRISPR biosafety needs to be adhered to
carefully. Protecting the staff and those in contact is essential to minimize exposures to infectious and
other hazardous agents. Concerning emerging technologies, there is a need for understanding the
relevant implications of possible hazards and potential effects f routes and materials being used.
c. Working with Nucleic Acids
i. PCR
The PCR process was carried out to initiate the pairs of the nucleotides ad to synthesize complete
strands of the individual fragments. The products yielded clones.
ii. Purification
After the screening process, the ctrl genes having the color mutants from the N-methyl-N’-nitro-N-
nitrosoguanidine (NTG) were isolated yielding purified successive vegetative cycles with same color signs
having true homokaryotic genes.
iii. Gel Electrophoresis
The observed strains were treated with ultraviolet light. Isolation was undertaken after screening in
about 5.6 × 105 spores.
iv. Measuring Concentration
Using off-target gene editing such as this case aiming crtl gene is for unintended genetic modifications.
Usage of Cas9 guide RNA is essential towards recognition of the 20 bp DNA sequence; this controls the
overall concentration levels of the Cas9 sgRNA complex (Perez-Pinera et al., 2014).
v. Calculating Molar Concentration
Calculation of the gRNA is based on the entrance of the DNA template sequences; it can be entered as a
full sequence or short around 20 nt. The formula used to calculate the molar mass followed the
following formula;
(A * 329.21) + (U * 306.17) + (C * 305.18) + (G * 345.21) + 159.0
Where the letters A, U, C, and G are the nucleotide number while the T's are converted to U's.

The optional options for the gRNA offer an avenue to calculated experiment volumes to allow targeting
DNA locations. These calculations assume the two RNA which require equal portion. The output volumes
are based on the one on one ratio of the Cas9 which is combined with the concentration of the gRNA
samples used.
d.Optical Density
Optical density is assessed based on the concentration estimate assessment at 260 nm, using this
finding, undertaking a calibration curve is essential for estimating the percentage f binding and release
of the Cas9/gRNA applied in the magnetic area (Kaushik et al., 2019).
e.Yeast Transformation
Usage of gRNA is estimated to yield a control transformation process that is geared towards repairing
the DNA. The introduction of the CRISPR in the plasmid introduces a repair template, repeated cleavage
causes toxicity. The transformation for the yeast takes place with the edition of the CRISPR-Cas9
protocol. Cas9 nucleus can create a double strand break at the choice locus which is lethal on the yeast
cells. This leads to efficiency repair of the homologous recombination process derived from the
polymerase chain reaction process (Akhmetov et al., 2018).

References
Akhmetov, A., Laurent, J.M., Gollihar, J., Gardner, E.C., Garge, R.K., Ellington, A.D., Kachroo, A.H. and
Marcotte, E.M., 2018. Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9. Bio-protocol,
8(6).
Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., Smith, I., Tothova, Z.,
Wilen, C., Orchard, R. and Virgin, H.W., 2016. Optimized sgRNA design to maximize activity and minimize
off-target effects of CRISPR-Cas9. Nature biotechnology, 34(2), p.184.
Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. and Joung, J.K., 2014. Improving CRISPR-Cas nuclease
specificity using truncated guide RNAs. Nature biotechnology, 32(3), p.279.
Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Adler, A.F., Kabadi, A.M., Polstein, L.R., Thakore, P.I., Glass,
K.A., Ousterout, D.G., Leong, K.W. and Guilak, F., 2013. RNA-guided gene activation by CRISPR-Cas9–
based transcription factors. Nature methods, 10(10), p.973.
Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Adler, A.F., Kabadi, A.M., Polstein, L.R., Thakore, P.I., Glass,
K.A., Ousterout, D.G., Leong, K.W. and Guilak, F., 2013. RNA-guided gene activation by CRISPR-Cas9–
based transcription factors. Nature methods, 10(10), p.973.
Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A. and Zhang, F., 2013. Genome engineering using
the CRISPR-Cas9 system. Nature protocols, 8(11), p.2281.