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How to Design Your gRNA for CRISPR Genome Editing

Posted by Guest Blogger on Sep 24, 2020 9:15:00 AM

Originally published May 3, 2017 and last updated Sep 24, 2020

This post was contributed by guest blogger, Addgene Advisory Board member, and Institute Scientist at the Broad Institute, John Doench.

CRISPR technology has made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post will discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's Validated gRNA Sequence Datatable. A more extended discussion of these subjects can be found in two recent review articles (Doench et al., 2017, and Hanna et al., 2020) and references therein.

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Topics: CRISPR, CRISPR gRNAs

CRISPR 101: Multiplex Expression of gRNAs

Posted by Mary Gearing on Sep 10, 2020 7:45:00 AM

Originally published Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang.

CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust system, editing or labeling efficiency doesn’t usually change when you add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!

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Topics: CRISPR, CRISPR 101, CRISPR gRNAs

Prime Editing: Adding Precision and Flexibility to CRISPR Editing

Posted by Jennifer Tsang on Oct 24, 2019 9:26:53 AM

Updated June 5, 2020.

There are over 75,000 pathogenic genetic variants that have been identified in humans and catalogued in the ClinVar database. Previously developed genome editing methods using nucleases and base editors have the potential to correct only a minority of those variants in most cell types. A new technique from David Liu’s lab at the Broad Institute could add more precision and flexibility to the CRISPR editing world.

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Topics: CRISPR, Cas Proteins, CRISPR gRNAs, Base Editing

Custom CRISPR Screens & the Green Listed Software

Posted by Guest Blogger on Jul 11, 2017 10:30:00 AM

This post was contributed by guest blogger Fredrik Wermeling, leader of a research group at the Centrum for Molecular Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden.

It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR screen. Luckily, the Green Listed software can help you do just this, probably in less than a minute (1).

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Topics: CRISPR, CRISPR gRNAs

Truncated gRNAs for Regulating Gene Expression

Posted by Guest Blogger on Jan 10, 2017 10:37:46 AM

This post was contributed by guest bloggers Alissa Lance-Byrne and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering.

CRISPR/Cas9 technology has revolutionized the fields of molecular biology and bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to either inactivate a gene or introduce a desired genetic alteration.

Listen to Our Podcast Intervew with Alex Chavez

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Topics: CRISPR, CRISPR gRNAs

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