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CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

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Identification of Circular RNAs using RNA Sequencing

Identification of Circular RNAs using RNA Sequencing

Published on: November 14, 2019

Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system.

Siqi Li¹, Hao Wu¹, Ling-Ling Chen^2,3,4

¹State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.

Nature Protocols

|July 13, 2022

Summary

This study introduces a CRISPR-Cas13 based protocol for large-scale screening of circular RNAs (circRNAs) in human cells. The method efficiently identifies circRNAs impacting cell growth without affecting messenger RNAs (mRNAs).

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Area of Science:

Molecular Biology
Genetics
RNA Biology

Background:

Circular RNAs (circRNAs) are key regulators of gene expression.
Distinguishing circRNA function from messenger RNA (mRNA) function is challenging due to sequence overlap.
The CRISPR-Cas13 system offers a potential solution for specific circRNA targeting.

Purpose of the Study:

To present a detailed protocol for large-scale functional circRNA screening using the RfxCas13d/BSJ-gRNA system.
To enable the identification of circRNAs that influence cell growth.
To provide a method that specifically targets circRNAs without affecting their cognate mRNAs.

Main Methods:

Utilizing a programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system with guide RNAs (gRNAs) targeting back-splicing junction (BSJ) sites.
Designing and constructing a gRNA library for efficient transduction.
Analyzing screening results and validating functional circRNA candidates in human cell lines.

Main Results:

The RfxCas13d/BSJ-gRNA system effectively and specifically discriminates circRNAs from mRNAs.
The protocol facilitates large-scale functional screening of circRNAs.
The method can identify circRNAs affecting cell growth under various conditions.

Conclusions:

The RfxCas13d/BSJ-gRNA protocol provides a robust method for circRNA functional screening.
This approach allows for the specific investigation of circRNA roles in cellular processes.
The protocol is applicable in both cellular and in vivo models for identifying functionally significant circRNAs.