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Comparing Copy Number Variations and SNPs

Comparing Copy Number Variations and SNPs

Sequencing of the human genome has opened up several best-kept secrets of the genome. Scientists have identified thousands of genome variations that exist within a population. These variations can be a single nucleotide or a larger chromosomal variation.
Copy number variations or CNVs are the structural variations that cover more than 1kb of DNA sequence. The single nucleotide polymorphism (SNP), on the other hand, is a single nucleotide change or a point mutation that is found in more than 1%...

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Updated: Dec 26, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Published on: February 26, 2019

Highly Parallel Profiling of Cas9 Variant Specificity.

Jonathan L Schmid-Burgk¹, Linyi Gao¹, David Li¹

¹Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

|March 19, 2020

Summary

This summary is machine-generated.

Researchers developed a new method to analyze Cas9 gene editing specificity. They identified LZ3 Cas9, a variant with high specificity and a unique insertion profile, revealing a trade-off between Cas9 activity and specificity.

Keywords:

CRISPR-Cas9 Cas9 specificity variants double-strand break detection off-target analysis tagmentation

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Published on: April 8, 2017

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Assessing the specificity of programmable nucleases like Cas9 is crucial for safe and effective genome editing.
Existing Cas9 variants aim to improve specificity, but comprehensive profiling across multiple targets is needed.
Understanding off-target cleavage and insertion profiles is essential for predicting and mitigating unintended mutations.

Purpose of the Study:

To introduce and validate tagmentation-based tag integration site sequencing (TTISS) as an efficient and scalable method for analyzing Cas9-induced double-strand breaks (DSBs).
To comprehensively compare the specificity and activity profiles of eight Cas9 variants across 59 genomic targets.
To identify novel Cas9 variants with enhanced specificity and activity, including those with unique insertion profiles.

Main Methods:

Development and application of tagmentation-based tag integration site sequencing (TTISS) for analyzing DSBs.
Parallel analysis of eight Cas9 variants across 59 distinct genomic targets using TTISS.
Generation and screening of thousands of additional Cas9 variants to identify improved specific and active versions.

Main Results:

Identified LZ3 Cas9, a high-specificity variant exhibiting a distinct +1 insertion profile.
Demonstrated a general trade-off between Cas9 activity and specificity across the tested variants.
Provided data on the frequency of +1 insertions, relevant for frameshift mutation correction strategies.

Conclusions:

TTISS is an efficient and scalable method for detailed analysis of Cas9 off-target cleavage and insertion profiles.
The identified LZ3 Cas9 variant offers high specificity and a unique insertion characteristic.
The study highlights a fundamental activity-specificity trade-off in Cas9 variants, informing future genome editing tool development.