The PAM… that sneaky little bit of sequence that you hope is present next to the "perfect" guide sequence for your genome engineering experiment. With CRISPR entering the clinic for correction of disease-causing alleles, and the growing need for gene editing in research, the old PAM constraints just won’t cut it anymore. Say hello to PAM-free and PAM-flexible nucleases! In this blog, we will review Cas enzymes, which have a flexible or non-existent PAM requirement, and how these proteins are advantageous in today’s genome editing landscape ..read more

Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers for gene targeting. Fast forward a decade, and the CRISPR field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it can be overwhelming to choose a Cas for your experiment. Even worse, you may end up missing an enzyme that is a better experimental fit due to this. But now, it’s time to cast aside those concerns and embrace a new resource to solve the problem: CasPEDIA, a new resource from Jennifer Doudna’s lab at the Innovative Genomics ..read more

Annotation of genes in immune cells typically involves the creation of germline knockout mice, which is time-consuming, as it only changes one gene at a time. CRISPR-based systems enable gene knockout in immune cells in a high-throughput manner, but these systems have not been widely employed in vivo. The CHIME and new X-CHIME systems, developed and deposited at Addgene by Arlene Sharpe’s lab, allow for wide deployment of in vivo gene knockout in immune systems.   ..read more

There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field and the greater scientific community. In 2014, we wrote a blog post on CRISPR’s potential for correcting monogenetic diseases. Now, almost 10 years later, CRISPR’s potential for treatment is no longer an accurate descriptor; progress in clinical therapy is more fitting, so it is time to update our post ..read more

Originally written by Marcy Patrick and Mary Gearing on Mar 12, 2015; updated by Christina Mork, Jul 27, 2020; updated by Susanna Stroik January 24, 2023. DNA damage drives genome instability and contributes to cancer, premature aging, and other harmful processes. The most dangerous type of DNA lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces such as ionizing radiation, chemotherapeutics, and ultraviolet light. In this post, we will describe the mechani ..read more

This post was originally written by Melina Fan and updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells with Cas9. Hooray! Now it’s time to make sure your genome edits went according to plan. In this blog post we’ll explain how to verify that your cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-in. We’ll also discuss what to do if your editing efficiency isn’t as great as you would like, you have options ..read more

This post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent of CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers to knock out, knock in, base edit, activate, repress, and even image your favorite gene. With so many Cas9 reagents available, it can be difficult to decipher which one is right for your unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s repository) and make it easy ..read more

The natural CRISPR locus of a bacteria host encodes multiple guide RNAs (gRNAs) on a single array to target the genome of the invading phage pathogen. Over the past decade, CRISPR tools have leveraged such host-defense mechanisms to enable multiplex gene editing in a variety of cells and organisms. However, lengthy genetic payloads and insufficient transcription on the array have limited the scalability and efficiency of multiplex gene editing. Moreover, existing multiplex strategies have been facing difficulties in pairing with base editing and prime editing approaches. Recently, Xue Sherry ..read more

This post was originally written in 2014 by Kendall Morgan and updated in 2022 by Lucie Wilson. Lucie is an Addgene co-op from Northeastern University.  There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field. It has the possibility to treat human genetic diseases, and several treatments are currently being tested in clinical trials. It is speeding the process of the discovery/development of genetic targets and RNA therapies. [6]. In 2014, we wrote a blog post on CRISPR’s potential for monogenetic diseases, the bulk of which you can find bel ..read more

The hardest part of any revolutionary discovery, including CRISPR, is turning potential into impact. In molecular and cellular biology, this happens through the development of tools that exploit and expand our current knowledge. The Abudayyeh-Gootenberg lab (also called the AbuGoot lab), a joint lab run by Omar Abudayyeh, PhD, and Jonathan Gootenberg, PhD, focuses exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two tools that expand and exploit CRISPR, both of which we're featuring in today's blog ..read more