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CRISPER Gene Therapy Recent Trends and Clinical Applications
Published in Yashwant Pathak, Gene Delivery, 2022
Prachi Pandey, Jayvadan Patel, Samarth Kumar
In the expression and maturation phase, the leader sequence positioned upstream to the CRISPR loci acts as a promoter and initiates transcription of the loci, producing long precursor CRISPR RNA or pre-crRNA. Following this step, processing of this pre-crRNA into small mature units, referred as crRNA takes place [38, 41, 42]. Representation of crRNA is exhibited by the joining of a spacer segment (sequence present complementarily to the foreign nucleic acid) at the 5′ end to repeat segment at the 3′ end [43, 44].
Ethics of Medical Product Development
Published in Howard Winet, Ethics for Bioengineering Scientists, 2021
As of this writing, this case has not exhausted appeals. The CRISPR-Cas9 system was originally developed for gene editing of prokaryotic cells by Jennifer Duodna of the University of California, and her colleagues (Jinek et al. 2012). Recall from Chapter 11 that during the adaptive immunity phase of a CRISPR-forming bacterium’s response to an attacking bacteriophage, a palindromic RNA code is transcribed to form a pre-CRISPR-RNA complex that joins with a CAS nuclease to produce a CRISPR-RNA-Cas complex. One particular Cas, Cas9 was found to be effective in finding the PAM and executing the required gene editing cut, by the Duodna group, and they filed to patent it for prokaryotes in 2012. Duodna stated then that the gap between prokaryotes and eukaryotes was significant enough to be a “large bottleneck” in the quest to transfer the technique to human cells (Sherkow 2018). Nevertheless, her group achieved the desired targeting the following year (Gilbert et al. 2013). Later that same year, Feng Zhang of the Broad Institute, Inc., and his group reported CRISPR-Cas9 system editing of eukaryotic cells (Ran et al. 2013). They had also filed a patent in 2012, for the system, after Duodna. Their patent differed in that it specified that their system targeted eukaryotes. The Zhang patent was approved first (Cockbain and Sterckx 2020).
Non-VLPs
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Abudayyeh et al. (2016) further showed that the novel CRISPR-Cas effector C2c2, now classified as Cas13a (Shmakov et al. 2017), which was developed from the type-VI CRISPR/Cas adaptive immune system of the bacterium Leptotrichia shahii, demonstrated RNA-guided ribonuclease function. The C2c2 could therefore provide interference against RNA phage. The in vitro biochemical analysis showed that the C2c2 was guided by a single crRNA, a CRISPR RNA, and could be programmed to cleave single-stranded RNA targets carrying complementary protospacers. Moreover, coexpression of the C2c2 and a crRNA containing a 28-nucleotide spacer targeting the phage MS2 RNA conferred viral resistance to E. coli. To validate the interference activity of the enriched spacers, the four top-enriched spacers were cloned individually into the pLshC2c2 CRISPR arrays and observed a 3- to 4-log10 reduction in plaque formation. The 16 guides targeting distinct regions of the MS2 maturation gene were cloned. All 16 crRNAs mediated MS2 interference, indicating that the C2c2 can be effectively retargeted in a crRNA-dependent fashion to sites within the MS2 genome (Abudayyeh et al. 2016). This remarkable finding was reviewed operatively by Zhang Dandan et al. (2016).
Critical insight into recombinase polymerase amplification technology
Published in Expert Review of Molecular Diagnostics, 2022
RPA is also being utilized as preamplifier in the two emerging CRISPR-based diagnostic technologies: SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter) [43]. With the discovery of the collateral cleavage activity of Cas12 and Cas13 proteins, several CRISPR-based rapid nucleic acid detection technologies have been developed. CRISPR-based technologies SHERLOCK and DETECTR utilize Cas13 and Cas12, respectively. In these technologies, CRISPR RNA (crRNA) is designed complementary to the target sequence, so that the crRNA will bind with the target with high specificity and activate the Cas12 or Cas13 protein. The activated Cas12 or Cas13 will cleave the target and also chop the neighboring fluorophore-labeled oligonucleotide probe through the nonspecific collateral cleavage activity. To improve sensitivity, the SHERLOCK and DETECTR utilize RPA to amplify target prior to detection through CRISPR.
Diagnostic accuracy of CRISPR technology for detecting SARS-CoV-2: a systematic review and meta-analysis
Published in Expert Review of Molecular Diagnostics, 2022
Xin Li, Huiling Zhang, Jing Zhang, Yang Song, Xuening Shi, Chao Zhao, Juan Wang
In 2013, Zhang et al. have firstly reported CRISPR-Cas Systems used in multiplex genome engineering, and later CRISPR technology came to apply for developing detection technology [8,9]. Diagnostic technology based on CRISPR-Cas can simultaneously satisfy a variety of detection criteria, which has the potential to be used to the next generation of diagnostic technology [10,11]. CRISPR Cas system can edit target DNA or RNA sequences with CRISPR Cas enzymology and amplification process in disease diagnosis platforms. Firstly, the vast amount of target nucleic acids synthesized with an amplification process, such as RT-RPA (reverse transcription recombinase polymerase amplification) or RT-LAMP [12]. Secondly, CRISPR RNA (crRNA) identifies the target nucleic acid for the spacer of the CRISPR Cas system and then the target nucleic acid was cut by specific Cas nucleases including Cas 9, Cas12 and Cas13 nucleases [12–14]. Finally, target nucleotides testing result was output by signal readout, such as fluorescence detection, lateral flow assay (LFA), and colorimetric aggregation with nanoparticles [12,15,16]. A large number of studies have reported that CRISPR technology has the advantages of low cost, ease to use, high sensitivity and specificity. To a certain extent, it can overcome the limitations of traditional molecular diagnostic methods such as RT-qPCR [17–19].
Restoration of dystrophin expression and correction of Duchenne muscular dystrophy by genome editing
Published in Expert Opinion on Biological Therapy, 2021
Tejal Aslesh, Esra Erkut, Toshifumi Yokota
Upon transcription, the CRISPR array produces pre-CRISPR RNA (pre-crRNA) which binds to the tracrRNA, producing mature crRNAs [14]. The endonuclease Cas9 can then recognize the guide RNA (gRNA) and can cleave the DNA sequence by inducing blunt double-stranded breaks (DSBs). A user-defined gRNA guides the Cas9 endonuclease to its target: a 20 nucleotide sequence (complementary to crRNA), which is adjacent to a PAM sequence. The PAM recognized by Cas9 is NGG or NAG (N = A, C, G, or T). Cas9 derived from Streptococcus pyogenes (SpCas9) is the most commonly used endonuclease. It cleaves at exactly 3 nucleotides from the PAM in the 3ʹ direction [10]. The bacterium Staphylococcus aureus-derived Cas9 (SaCas9) recognizes NNGRRT as a PAM sequence, limiting its potential targets for editing [15]. CRISPR/SpCas9 components are bulky and difficult to package into the common adeno-associated virus (AAV) vector (~4.85 kb limit). To overcome packaging issues, SaCas9 is preferred over SpCas9 owing to its smaller size (~1 kb smaller) [16].