From the battleground of bacteria and archaea against virus and plasmid DNA (pDNA), the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated (Cas) gene system gives an efficient and promising toolbox for genetic engineering. [1][2][3][4][5] Compared with other genome editing technologies, such as transcription activator-like effector nucleases (TALEN) and zinc-finger nucleases (ZFN), the CRISPR/Cas system lowers the bar of conducting genome editing experiments because it does not require redesign of nucleases to recognize distinct target gene. [6] With only one decade of development, the CRISPR/Cas systems have emerged as a revolutionary engineering tool for sequence-specific genome modifications and have brought significant advances in gene therapy, developmental biology, cancer research, etc. [7] Currently, dozens of CRISPR/Cas-based therapeutics have entered clinical trials, paving the way for precision medicine. [8][9][10] The CRISPR/Cas systems can be classified into two categories (class 1 and class 2) according to the number of Cas proteins involved. [11] The class 1 systems contain multiple Cas subunits, while class 2 systems only contain a single Cas effector protein. [11] The simplicity and flexibility of type II (CRISPR/Cas9) and V (CRISPR/Cas12) systems from class 2 make them more suitable for genetic engineering. [12] Both CRISPR/Cas9 and CRISPR/Cas12 utilize their own single-guide RNAs (sgRNAs) and protospacer adjacent motifs (PAM) to recognize and bind the target gene. [2,13,14] After the formation of the R-loop, the RNA-bounded Cas protein undergoes conformational rearrangement, and induces DNA double-strand break (DSB). [15,16] Eventually, the DNA repair system in eukaryotic cells is activated to repair the cleaved DNA primarily by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways (Figure 1). NHEJ is usually considered as a highly effective but error-prone mechanism due to the direct ligation of the broken DNA. The ligation process is easy to introduce random insertions or deletions (indels) at the target loci. [17] Thus, NHEJ repair is widely employed in treating diseases caused by the overexpression of abnormal proteins, for instance, transthyretin amyloidosis. [6] The HDR pathway uses homologous DNA templates to repair DNA lesions. [18] As a result, diseases caused by loss-of-function mutations, such as hemophilia, phenylketonuria, and X-linked retinitis pigmentosa, can benefit from CRISPR/Cas-mediated HDR. [10,19] In addition to NHEJ and HDR, alternative mechanisms for DSB repair including homology-mediated end joining (HMEJ), microhomology-mediated end joining (MMEJ), and homology-independent targeted integration (HITI) were also developed to broaden the scope of CRISPR/Cas system. [20][21][22][23] Although CRISPR/Cas systems hold great potential for therapeutic applications, there are two major challenges that need to be addressed. [24] The first challenge is to minimize off-target effects. [25] The second one i...
We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent in vitro activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, FM09h, was highly active with IC50 of 0.3 μM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of FM09h remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of FM09h (10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).
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