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Organoid Technology for Basic Science and Biomedical Research
Published in Hyun Jung Kim, Biomimetic Microengineering, 2020
Szu-Hsien (Sam) Wu, Jihoon Kim, Bon-Kyoung Koo
CRISPR/Cas9 technology utilizes a bacterial adaptive defense mechanism made up of a complex of a dual RNA hybrid containing single-stranded CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) together with the Cas9 endonuclease. The crRNA contains 20 nucleotides complementary to a target sequence and together with the tracrRNA directs Cas9 endonuclease to target site. Then, Cas9 generates double-strand breaks on target DNA adjacent to the PAM (protospacer adjacent motif) site, which allows self- and nonself-determination (Jiang and Doudna 2017). Using knowledge gained from basic scientific research into microbial defense mechanisms, engineering of the dual-tracrRNA:crRNA hybrid into a single molecule (termed single-strand guide RNA or gRNA) directing Cas9 to cleave double-stranded DNA (Jinek et al. 2012) has allowed gene targeting in various model systems through simply designing the 20 nucleotides of the gRNA sequence to match the desired genomic locus.
Microbial Biofilms-Aided Resistance and Remedies to Overcome It
Published in Bakrudeen Ali Ahmed Abdul, Microbial Biofilms, 2020
Currently, the CRISPR/Cas system id divided into three types. Out of them. Type II CRISPR/Cas system is the most studied one which differs phylogenetically and only found in bacteria (Hedge et al. 2019). This system makes use of trans-activating RNA (tracrRNA), CRISPR RNA (crRNA), and an endonuclease Cas 9, obtained from the type II system. TracrRNA has sequences complementary to crRNA, this chimeric form is known as single guide RNA (sgRNA). The sgRNA directs Cas9 to genomic DNA at 5′ end and binds to specific sequences called protospacer adjacent motif (PAM) typically NGG sequences. Five nucleotides upstream of PAM sequences mark the target site and is required for the double-stranded endonucleases activity of Cas 9 (Chaterji et al. 2017).
Development of capability for genome-scale CRISPR-Cas9 knockout screens in New Zealand
Published in Journal of the Royal Society of New Zealand, 2018
Francis W. Hunter, Peter Tsai, Purvi M. Kakadia, Stefan K. Bohlander, Cristin G. Print, William R. Wilson
From there the race was on to determine the mechanism by which CRISPR arrays might provide immunity against invading nucleic acids. In short order, it was shown that the CRISPR-associated Cas9 gene plays a critical role in the acquisition of phage resistance, that the gene encodes a nuclease capable of inducing double-strand breaks in DNA, and that immunity depends on precise DNA sequence identity between spacers and targets. There are many variants on the theme, but the operation of the simplest of the types of CRISPR-Cas system is illustrated in Figure 1. In this case, the CRISPR array is transcribed and then cut to generate CRISPR RNAs (crRNA) that act in concert with a second RNA (trans-activating CRISPR RNA; tracrRNA) to guide Cas9 to a sequence complementary to the crRNA where it functions like a specifically targeted restriction enzyme. The cut produces a blunt-ended double-strand break 3 nucleotides upstream of a 2–5 nucleotide sequence known as the protospacer adjacent motif (PAM). The latter sequences are absent from the palindromic repeats in the CRISPR array, precluding cutting of the array itself and thus contributing to self/non-self discrimination.