Details

Autor(en) / Beteiligte

• Schmid-Burgk, Jonathan L.
• Gao, Linyi
• Li, David
• Gardner, Zachary
• Strecker, Jonathan
• Lash, Blake
• Zhang, Feng

Titel

Highly Parallel Profiling of Cas9 Variant Specificity

Ist Teil von

• Molecular cell, 2020-05, Vol.78 (4), p.794-800.e8

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2020

Quelle

MEDLINE

Beschreibungen/Notizen

• Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations. [Display omitted] •Tagmentation-based tag integration site sequencing (TTISS) scalably detects DSBs•TTISS is a rapid and streamlined protocol compatible with multiplexing•Application of TTISS highlights trade-off in Cas9 variant specificity and activity•LZ3 Cas9 variant exhibits a unique +1 insertion profile Schmid-Burgk et al. develop tagmentation-based tag integration site sequencing (TTISS), a rapid, streamlined protocol for analyzing double-strand breaks such as those created by CRISPR nucleases. Using TTISS, they comprehensively assess Cas9 variants, revealing a trade-off between specificity and activity and identifying LZ3 Cas9, a variant with a unique +1 insertion profile.