Pam-less Editing
Pam-less Editing - Related Videos
02:23
RNA Editing
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2020
RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
08:31
Genome Editing
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2023
A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be...
02:58
CRISPR-Mediated Base Editing Tools: A Genome Editing Technique to Induce Targeted Base Substitution
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2025
This video explains the concept of CRISPR-mediated cytosine base editors for inducing targeted nucleotide substitution.
08:50
A Nonsequencing Approach for the Rapid Detection of RNA Editing
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2022
Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (µTGGE) as a simple, quick, and portable method of detecting RNA editing.
02:30
A CRISPR-Cas9 Technique for Gene Editing in T Cells
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2025
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell's non-homologous end-joining mechanism, leading to gene knockout.
FAQs
By no longer limiting target selection to sequences adjacent to a canonical PAM, PAM-less editing gives researchers more candidate sites within a genomic region. This broader search space can make it easier to position a guide over a pathogenic variant, regulatory element, or other sequence of interest, potentially improving the practicality of experimental design.
The outcome depends on the engineered or naturally PAM-flexible CRISPR-associated protein and the editor configuration used with its guide RNA. Some systems are associated with DNA cleavage, whereas others perform targeted base conversion. Selecting between these modes connects the molecular mechanism to whether a study seeks broader sequence disruption or a specific nucleotide correction.
Access to more genomic sites does not by itself guarantee precise editing. Because the approach can reach previously inaccessible regions, researchers must assess whether the selected guide and protein act at the intended sequence and whether unintended edits occur elsewhere. This evaluation is important when interpreting gene-function experiments and considering therapeutic correction, where inaccurate changes could alter biological conclusions.
Conventional CRISPR targeting is limited by the availability of a suitable canonical PAM near the desired sequence, while PAM-less editing uses proteins with greater PAM flexibility. The practical difference is access: sites excluded by the canonical requirement may become targetable, expanding possible guides and editing locations. The tradeoff is the need for careful specificity assessment rather than assuming broader reach ensures better performance.
A conceptual workflow begins by identifying the genomic sequence that could not be readily targeted under canonical PAM rules, then selecting a compatible PAM-flexible CRISPR-associated protein and guide RNA. Researchers next choose an editor suited to the intended outcome, such as cleavage or base conversion, and evaluate specificity and unintended edits. These decisions connect target accessibility with experimental interpretation.
Expanded target access can support studies of pathogenic variants, regulatory elements, and other genomic regions that were previously difficult to reach. In biology, this enables researchers to examine how selected sequences influence gene function and investigate whether targeted correction is feasible. The value lies not only in reaching a site, but also in linking the edit to a measurable biological question.
Researchers can compare whether a chosen system reaches the intended genomic site and whether its activity produces cleavage or targeted base conversion. They should also consider specificity and unintended edits when interpreting results. Comparing these outcomes helps distinguish successful access from useful biological editing, especially in experiments focused on gene function, variant analysis, or possible therapeutic correction.