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CRISPR 101

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CRISPR 101

CRISPR 101: Any Base Transversion Editors

Emily P. Bentley April 1, 2025

In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs first convert a cytosine (C) to uracil (U), just like Cytosine Base Editors (CBEs). But unlike CBEs, CGBEs then excise the U to create an abasic (empty) DNA site using ...
The nucleobases are shown with arrows describing conversions between them. In step one, an adenosine deaminase converts adenine to hypoxanthine (the nucleobase component of inosine); this is catalyzed directly by the base editor. Next, base excision of hypoxanthine (also by the base editor) could be repaired in two different ways by the cell, shown by an arrow that splits into two outcomes. The repair pathway leading to cytosine is favored by ACBEs, while the repair pathway leading to thymine is favored by AYBEv3 + Polη.
CRISPR 101

CRISPR 101: Cytosine Transversion Editors

Emily P. Bentley March 25, 2025

The first base editors revolutionized CRISPR gene editing. Cytosine base editors (CBEs) and adenine base editors (ABEs) chemically modify target bases without breaking the DNA backbone, making them efficient and precise tools for altering DNA sequences. These first base editors ...
The nucleobases are shown with arrows describing conversions between them. In step one, a cytosine deaminase converts cytosine to uracil; this is catalyzed directly by the base editor. Next, base excision of uracil (also by the base editor) is repaired in two different ways by the cell, shown by an arrow that splits into two outcomes. Repair in E. coli leads to adenine, while repair in mammalian cells leads to guanine.
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

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

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

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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