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Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

Posted by Mary Gearing on Apr 5, 2018 8:42:28 AM

CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including the standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka groups have obtained an average knock-in efficiency of 30-60%. This is much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR mouse model generation easi-er!

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Topics: CRISPR, Cre-lox

xCas9: Engineering a CRISPR Variant with PAM Flexibility

Posted by Mary Gearing on Mar 28, 2018 2:52:13 PM

In order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes, but a PAM is not always available near the locus you’d like to modify. To tackle the PAM problem, researchers have engineered alternative Cas9s binding distinct PAM sequences. Now, Hu et al., working in David Liu’s lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more about xCas9 - here’s what we know so far!

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Topics: CRISPR

CRISPR 101: Cas9 Nickase Design and Homology Directed Repair

Posted by Mary Gearing on Mar 15, 2018 8:59:40 AM

By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent gRNAs, can lower the probability of off-target editing. But that’s not all! New research from IDT (Integrated DNA Technologies) has shown that a nickase approach can improve homology directed repair (HDR) rates, provided you follow some simple design rules described below.

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Topics: CRISPR, CRISPR 101

CRISPR Challenges: Standardization and Homology Directed Repair

Posted by Mary Gearing on Feb 14, 2018 9:36:24 AM

Blugene and I represented Addgene at the recent Keystone meeting on Precision Genome Editing with Programmable Nucleases. Check out #KSgenome on Twitter if you missed our live updates!

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Topics: CRISPR

Genome Engineering, CRISPR, and Research in Singapore: Interview with Wei Leong Chew

Posted by Tyler Ford on Feb 6, 2018 9:00:56 AM

In today’s podcast, we sit down with Wei Leong Chew, a researcher at the Genome Institute of Singapore who recently started his own lab. We discuss some of the joys and difficulties of getting a lab up and running, and learn a little bit about what it was like for Wei Leong to work in George Church’s lab as a graduate student.

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Topics: Genome Engineering, CRISPR, Podcast

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