Titlebook: Recombineering; Methods and Protocol Christopher R. Reisch Book 2022 Springer Science+Business Media, LLC, part of Springer Nature 2022 CRI

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Proponent

Genome Editing in , Using CRISPR/Cas9 Technology,tem as well as a cytidine deaminase-mediated base editing system (pBECKP) in ., enabling rapid, scarless, and efficient genetic manipulation in diverse . strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in ..

COMA

Genome Editing of , Using CRISPR-Cpf1 System,icroorganisms but cannot work well in .. We recently developed two . (.) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in .. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 . + 4 or 3 . + 2 days, respectively.

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CRISPR/Cas-Mediated Genome Editing of ,rious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of .. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.

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outset

Genome Editing Methods for , needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.

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crucial

Dup-In and DIRex: Techniques for Single-Step, Scar-Free Mutagenesis with Marker Recycling, selection for spontaneous loss of the counter-selectable cassette, which leaves only the intended mutation behind in the final strain. The techniques have been used in several strains of . and ., and should be readily adaptable to other organisms where λ Red recombineering or similar methods are available.

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Christopher R. ReischIncludes cutting-edge methods and protocols.Provides step-by-step detail essential for reproducible results.Contains key notes and implementation advice from the experts

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