Small indels induced by CRISPR/Cas9 in the 5′ region of microRNA lead to its depletion and Drosha processing retardance

Qian, Jiang; Meng, Xing; Meng, Lingwei; Chang, Nannan; Xiong, Jing-Wei; Liang, Zicai

doi:10.1080/15476286.2014.996067

Cited by 34 publications

(31 citation statements)

References 25 publications

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“…Recent studies reported the successful knockout of miRNA genes using TALEN and Cas9 nucleases. 4,22,23 We used a TALEN-based knockout technique to specifically delete miR-221 and miR-222 (paralogous miRNAs), which did not noticeably affect expression of the other miRNA (Fig. 2B).…”

Section: Discussionmentioning

confidence: 99%

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

et al. 2017

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MicroRNAs (miRNAs) regulate the expression of mRNA through sequence-specific binding of the 3 0 untranslated region (UTR). The seed sequence of miRNAs is the key determinant for target site recognition. Paralogous miRNAs, which share the same seed sequences but differ in their 3 0 regions, are known to regulate largely overlapping groups of mRNAs. However, no study has analyzed functional differences between paralogous miRNAs with proper experimental methods. In this study, we compared the targets of paralogous miRNAs, miR-221 and miR-222. Using a nuclease-mediated genome engineering technique, we established knockout cell lines for these miRNAs, and precisely analyzed differences in target regulation. We found that miR-221 and miR-222 suppress the previously identified targets, CDKN1B and CDKN1C, differentially. Whereas both miRNAs suppressed CDKN1B, only miR-221 suppressed CDKN1C. From transcriptome analyses, we found that several different target mRNAs were regulated by each of miR-221 and miR-222 independently, although a large number of mRNAs responded commonly to miR-221 and miR-222. This is the first study to compare the mRNA regulations by paralogous miRNAs and illustrate that paralogous miRNAs with the same seed sequence also have difference in target regulation.

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Section: Discussionmentioning

confidence: 99%

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

et al. 2017

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“…NHEJ often results in insertions or deletions (indels) at the cleavage site, which can cause frame shift mutations in protein-coding genes. MicroRNA genes can also be inactivated by the formation of indels in the mature miRNA sequence or in regions necessary for the biogenesis of the miRNA (Jiang et al 2014;Chen et al 2015a;Chang et al 2016).…”

Section: Introductionmentioning

confidence: 99%

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs

Kurata

Lin

2018

RNA

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MicroRNAs (miRNAs) are post-transcriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however, few libraries have included miRNA genes. Here we constructed a miRNA-focused CRISPR-Cas9 library that targets 1594 (85%) annotated human miRNA stem-loops. The sgRNAs in our LX-miR library are designed to have high on-target and low off-target activity, and each miRNA is targeted by four to five sgRNAs. We used this sgRNA library to screen for miRNAs that affect cell fitness of HeLa or NCI-N87 cells by monitoring the change in frequency of each sgRNA over time. By considering the expression in the tested cells and the dysregulation of the miRNAs in cancer specimens, we identified five HeLa pro-fitness and cervical cancer up-regulated miRNAs (miR-31-5p, miR-92b-3p, miR-146b-5p, miR-151a-3p, and miR-194-5p). Similarly, we identified six NCI-N87 pro-fitness and gastric cancer up-regulated miRNAs (miR-95-3p, miR-181a-5p, miR-188-5p, miR-196b-5p, miR-584-5p, and miR-1304-3p), as well as three anti-fitness and down-regulated miRNAs (let-7a-3p, miR-100-5p, and miR-149-5p). Some of those miRNAs are known to be oncogenic or tumor-suppressive, but others are novel. Taken together, the LX-miR library is useful for genome-wide unbiased screening to identify miRNAs important for cellular fitness and likely to be useful for other functional screens.

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“…Functional disruption of the miR‐744 precursor sequence can be achieved either by inserting a point mutation, which is also very effective for protein encoding genes by disrupting the open reading frame, or by excising the entire gene. Although point mutations targeting either the hairpin‐loop or the 5′ seed region of miRNAs were reported for human cancer cell lines, our excision‐strategy to eliminate the entire miRNA gene allowed for simple and fast screening via PCR amplification of the target locus after FACS of GFP‐positive single cells. Low screening effort in parallel to high editing efficiency was assured by encoding both 5′ and 3′ targeting sgRNAs in a tandem array together with Cas9‐T2A‐GFP on a single expression vector .…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

Raab

Mathias

Alt

et al. 2019

Biotechnology Journal

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MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L −1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L −1 , pointing towards the potential of miRNA KO for cell line engineering.

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Small indels induced by CRISPR/Cas9 in the 5′ region of microRNA lead to its depletion and Drosha processing retardance

Cited by 34 publications

References 25 publications

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

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