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CRISPR (2)

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CRISPR

Typing CRISPR Systems

Alyssa Shepard March 18, 2025

What’s in a type? That which we call CRISPR, by any other name would…probably still edit genomes.
A schematic overview of the CRISPR classification system.
CRISPR

CRISPR 101: Cytosine and Adenine Base Editors

Multiple Authors February 13, 2025

Early CRISPR applications were often limited by the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end joining (NHEJ), HDR occurs at a relatively low ...
A cartoon depiction of cytidine base editing. A base editor, consisting of a cytidine deaminase fused to Cas9, is shown binding to DNA using its guide RNA. The guide RNA base pairs to target DNA, leaving the opposite strand of DNA free to be contacted by the cytidine deaminase, which converts a C to a U within this single-stranded sequence. This deamination yields DNA with a G:U mismatch without creating a double-strand break. Mismatch repair preserves the edit IF the modified strand is used as the template, converting the mismatched G to an A and yielding a single-base-pair edit.
CRISPR

Degrading DNA with Cascade-Cas3

Alyssa Shepard February 11, 2025

The versatility of CRISPR allows you to play with DNA in a number of ways, from small edits that change single base pairs, to chromosomal inversions and large deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding repertoire of CRISPR has ...
Diagram of the Cascade complex with gRNA bound to target DNA, after recruitment of Cas3. Cas3 is about to nick the non-target strand.
CRISPR

The AAVantages of AAV in CRISPR Screens

Alyssa Shepard February 4, 2025

Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target genes for drug discovery or to understand the intricacies of molecular pathways. These screens have gotten larger, easier, and more comprehensive thanks to the consistent ...
Graphic showing the workflow of using a pooled AAV CRISPR library in vitro. Step A shows the AAV containing the library with either the gRNA only or gRNA plus Cas9 and infecting cells expressing Cas9 or wild-type. The different guides are represented by different colors in individual cells. Step B shows the results of the positive or negative selection, with only one type of guide (color) being chosen. Step C shows verification using next generation sequencing.
CRISPR

Design Tips for Prime Editing

Emily P. Bentley January 23, 2025

We recently updated our blog post on Prime Editing, and that meant rereading many of the original papers reporting various prime editing tools. These papers are chock full of great tips to guide your experimental design, especially the design of the RNA sequences you’ll use in ...
A cartoon of a prime editor with two different edit sequences. The DNA sequences are shown with one strand edited and a 5′ DNA flap, before heteroduplex resolution and DNA repair. The first edit has an unchanged PAM. This DNA is shown connected to the prime editor by a two-way arrow, indicating that the editor can re-bind. Re-nicking is represented by scissors and would remove the newly edited DNA. The second edit has an altered PAM. A one-way arrow leads from the prime editor to this edit, indicating that the changed PAM prevents the editor from re-binding.
CRISPR

CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

Alyssa Neuhaus November 14, 2024

You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest of our genome could possibly be made up of. The answer is… drum roll please… non-coding RNAs! You probably didn’t see that coming, right? Non-coding RNAs ...
Alt text: A schematic illustration of the CRISPR/Cas9-based approaches mentioned above. The first illustration (A) shows a DSB made at a transcription start site upstream of an exon. The second illustration (B) shows a DSB made at splice sites. The third illustration (C) shows a DSB made upstream and downstream of an exon to fully remove a genomic fragment. The fourth illustration (D) shows a DSB made within an exon to insert a synthetic polyadenylation signal. 
CRISPR

CRISPR Guide Update

Alyssa Shepard October 10, 2024

Addgene is proud to present our updated CRISPR Guide!
CRISPR prime editing schematic.
CRISPR

Making CRISPR Plasmids Using Fragmid

Rachel Leeson August 29, 2024

If you’ve ever been looking for just the right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close enough, you’ve found yourself with a common dilemma. Request the vectors you can find and use them as-is, saving time and effort but risking ...
Schematic of Fragmid assembly process from day one to day four and resulting vector architecture. On day one fragment plasmids plus a destination vector are assembled using Golden Gate (BbsI). On day two the assembled vector containing a Guide (2xBsmBI), Promoter, N-terminus, Cas protein, C’terminus, and 2A-Selection undergoes an exonuclease V cleanup followed by transformation and plating. On day three two colonies per construct are picked, miniprepped and restriction digested for gel validation/whole plasmid sequencing. Below the assembled vector are examples of six vector architectures including pRDA_512 lentivirus, pRDA_722 lentivirus, pRDA_789 CROPseq lentivirus, pRDA_889 AAV, pRDA_575 Piggyback, and pRDA_791 empty plasmid.

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