The Broad Institute of Harvard and MIT David Liu and Boston UniversityAhmad S. Khalil and other researchers used directed evolution strategies to filter the -compatible PAM (protospacer adjacent motif) sequences that can be used to edit single bases for . Functional small Cas protein. The characteristic of this directional evolution strategy is that considers the binding to PAM sequence and the function of single-base editing ; it mainly integrates the functional Cas protein screening of PAM recognition , phage-assisted high-throughput automated bacterial culture control screening Platform and fast PAM sequence compatibility analysis and other technologies (1–4).
Highly compatible PAM sequence Cas protein orientation evolution screening strategy design concept (1)
PAM recognition functional Cas protein screening (1)
phage-assisted high-throughput automated bacterial culture control screening system (1)
Researchers' researcher's targeting Evolutionary technology screened out , a small Cas protein variant with better compatibility with multiple PAM sequences of , and has higher efficiency or lower off-target effects in certain scenarios of ( 1).
This work was published in nature biotechnology on September 8, 2022; researchers said that more compatible PAM sequences will allow single-base editing tools to cover a wider range of disease mutation tools (1).
Comment(s):
Universal After all, highly controllable culture and high-throughput acquisition of bacterial culture status data, it can also be used to optimize other gene editing/manipulation systems, etc.
In addition, further engineered bacteria to design to integrate potential off-target effects ; or combines directional evolution, structural analysis and rational design of , which is expected to efficiently obtain more flexible and practical Cas variants.
References:
1. T. P. Huang et al., High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs. Nat. Biotechnol. 2022, 1–12 (2022).
2. B. G. Wong, C. P. Mancuso, S. Kiriakov, C. J. Bashor, A. S. Khalil, Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nat. Biotechnol. 2018 367. 36, 614–623 (2018).
3. A. H. Badran, D. R. Liu, Development of potential in vivo mutagenesis plasmids with broad mutational spectra. Nat. Commun. 2015 61. 6, 1–10 (2015).
4. J. H. Hu et al., Evolved Cas9 variants with broad PAM compatibility and high DNA specification. Nature. 556, 57–63 (2018).
Original link:
https://www.nature.com/articles/s41587-022- 01410-2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-