Cleavage of DNA Substrate Containing Nucleotide Mismatch in the Complementary Region to sgRNA by Cas9 Endonuclease: Thermodynamic and Structural Features

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(19), С. 10862 - 10862

Опубликована: Окт. 9, 2024

The non-ideal accuracy and insufficient selectivity of CRISPR/Cas9 systems is a serious problem for their use as genome editing tool. It important to select the target sequence correctly so that system does not cut similar sequences. This requires an understanding how why mismatches in can affect efficiency Cas9/sgRNA complex. In this work, we studied catalytic activity Cas9 enzyme cleave DNA substrates containing nucleotide mismatch at different positions relative PAM "seed" sequence. We show complementarity sgRNA/DNA duplex protospacer adjacent motif (PAM) tend decrease cleavage increase half-maximal reaction time. However, two 11 20 PAM, was observed, both with without half-reaction Thermodynamic parameters were obtained from molecular dynamics results, which showed 8, 11, thermodynamically stabilize formed complex, position 2 fragment exerts greatest stabilization compared original weak correlation thermodynamic binding components Cas9/sgRNA:dsDNA complex data indicates operation mainly affected by conformational changes mutual arrangement sgRNA substrates.

Язык: Английский

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Rational Design of Enhanced Nme2Cas9 and Nme2^SmuCas9 Nucleases and Base Editors

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Окт. 30, 2024

Abstract CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced editors are Cas9 nucleases, but many nuclease technologies provide only limited control over outcomes. Adenine base (ABEs) and cytosine (CBEs) precise efficient nucleotide conversions A:T-to-G:C C:G-to-T:A pairs, respectively. Therapeutic use (BEs) provides an avenue to correct approximately 30% human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, delivery limit the full therapeutic potential BEs. We previously developed Nme2Cas9 its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part near N 4 CC PAMs. Further engineering yielded domain-inlaid BEs enhanced activity, well Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N C) Here we further enhance Nme2 Smu effectors for improved efficiency compatibility through site-directed mutagenesis deaminase linker optimization. Finally, define specificity profiles resulting variants by using paired guide-target libraries.

Язык: Английский

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Unwinding matters: WED domain determines Cas9 activity by accelerating DNA unwinding and R‐loop formation

MedComm – Biomaterials and Applications, Год журнала: 2024, Номер 3(4)

Опубликована: Дек. 1, 2024

Язык: Английский

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