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Design Tips for Prime Editing

By Emily P. Bentley

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CRISPR

Prime Editing: Adding Precision and Flexibility to CRISPR Editing

Multiple Authors January 13, 2025

Over 75,000 pathogenic genetic variants have been identified in humans and cataloged 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. But ...
A cartoon overlayed on several crystal structures showing the parts of the prime editor: the Cas9 nickase domain, the reverse transcriptase domain, and the pegRNA.
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.
CRISPR

Build Your CRISPR Vocabulary

Emily P. Bentley July 2, 2024

CRISPR is a sleek acronym for a real mouthful of a phrase: Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity and complexity is reflected in the biology, too. CRISPR is an elegant bacterial immune system and an efficient gene editing tool… but ...
Overview of the parts of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both the target and nontarget DNA strands upstream of the PAM site found in the nontarget strand.
CRISPR

PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

Guest Blogger June 25, 2024

Prime editing is a versatile genome editing technology that allows precise modifications of DNA (replacements, small insertions, and deletions) without introducing DNA double-strand breaks (Anzalone et al., 2019; Chen & Liu, 2023). This method uses a prime editor (typically ...
screenshot of various PRIDICT webpages
CRISPR

A Needle in a Base-Stack: Cas9 Structural Biology

Emily P. Bentley June 4, 2024

Have you ever designed a CRISPR guide RNA and wondered why it is limited to only 20 bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent motif (PAM)? Cas9 is becoming an ever more ubiquitous tool for genome engineering, and studying its ...
Cartoon summary of Cas9 activity.

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