![]() ![]() However, this method led to unintended cleavage of targets carrying partially similar or complementary sequences, especially when the complementarity occurred in the nucleating "seed" region of the siRNA, while being incompatible with eukaryotic model systems. In the past, scientists had developed targeted RNA knockdown techniques in eukaryotes by using RNA interference or RNAi, whereby small interfering RNA-directed argonaute nucleases (the active part of the RNA-induced silencing complex) cleaved complementary target RNAs. Molecular biologists seek to alter RNA and protein levels without permanently affecting DNA however, the task is nontrivial in basic research and in therapeutic applications. Gene editing: From RNA interference methods to CRISPR-Cas complexes By catalytically inactivating the Csm, the team achieved durable RNA binding for live-cell RNA imaging to establish the efficiency of the CRISPR-Cas effector system as RNA-targeting tools in eukaryotes. The vector-bound Steptococcus thermophilus Csm-complex provided high-efficiency RNA knockdown a method to silence gene expression with minimal off-target impact in human cells and outperform existing genome editing technologies such as short-hairpin RNA and Cas13-mediated knockdown. Thereafter, the molecular biologists accomplished surgical RNA ablation (deletion) of the nuclear and cytoplasmic transcripts via single-vector delivery. The team used the clustered regularly incorporated short palindrome repeats (CRISPR)-Csm complex which included a protein known as Csm, a type III-A CRISPR-Cas interference complex found in the prokaryotic immune system. In a recent report now published in Nature Biotechnology, David Colognori and a research team headed by Chemistry Nobel Laureate Jennifer Doudna, who discovered and expanded on the CRISPR-Cas9 technology alongside Emmanuel Charpentier, in the year 2020, incorporated a new complex to the CRISPR complex during this study. Mammalian cells are inherently complex due to subcellular compartments, thereby making the process of robust transcript targeting of nucleic acids somewhat challenging in the molecular biology lab. l, Relative GFP fluorescence of HEK293T-GFP cells transfected with the indicated delivery vectors and expressing the indicated GFP-targeting crRNAs, measured by flow cytometry. One of two replicates with similar results is shown. GAPDH is shown as loading control (green). (k) Western blot showing proper size and expression of Cas/Csm proteins (red) in HEK293T cells. (j) Diagram showing all-in-one delivery vector designs. RFP-targeting crRNA is listed in Supplementary Table 1 of the paper. (i) Relative GFP and RFP fluorescence of HEK293T-GFP/RFP cells transfected with plasmids expressing Cas6, Csm1-5 and the indicated crRNAs (individual or multiplexed), measured by flow cytometry. h, Same as f, but with GFP crRNA 1 adjusted to the indicated spacer length. g, Same as f, but with the indicated Csm mutants (or crRNA + Cas6 only). (f) Relative GFP fluorescence (= MFI targeting crRNA/MFI nontargeting crRNA) of HEK293T-GFP cells transfected with plasmids expressing Cas6, Csm1-5 and the indicated GFP-targeting crRNA, measured by flow cytometry. ![]() ![]() (e) Immunofluorescence showing expression and nuclear localization of Cas/Csm proteins in HEK293T cells. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) shown as loading control (green). Csm1 and Csm4 are less stable when expressed separately. (d) Western blot showing proper size and expression of Cas/Csm proteins (red) in HEK293T cells. (c) Close-up of crRNA:target binding, showing the 6-nt cleavage pattern. crRNAs are transcribed from the CRISPR array, processed by Cas6 and assemble with Csm proteins. thermophilus type III-A CRISPR-Cas locus. (a) Diagram showing cis- and trans-cleavage of Cas13. An all-in-one type III CRISPR-Cas system in human cells. ![]()
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