Abstract: The present disclosure provides methods for producing gene-edited cells free of gene-editing system molecules through the manipulation of prototrophy. Exemplary system molecules include those required for CRISPR editing techniques, such as plasmids and genes encoding Cas nucleases. The methods may employ constructs that temporarily disrupt prototrophy, the removal of which restores prototrophy. Also disclosed are gene-edited cells and populations of gene-edited cells comprising these constructs. The present methods and compositions may be used to achieve desired gene editing of a host cell in the absence of extraneous genetic material remaining from the genetic engineering technique itself.

Abstract: Provided herein are nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals as well as expression vectors and compositions comprising the same. Also provided herein are methods for assembling the nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals in a pooled and/or modular manner. Methods for using the nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals to facilitate multiplexed genomic editing of a host cell comprising said nucleic acid constructs are also provided herein.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in successive rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in an iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in successive rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in an iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in one or more rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in a pooled and/or iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Compositions and kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in one or more rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in a pooled and/or iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Compositions and kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in successive rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in an iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in one or more rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in a pooled and/or iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Compositions and kits for performing the methods are also disclosed.

Abstract: The present invention relates to methods for editing the genome of a microbial host cell in successive rounds of transformation. The method allows the introduction of genetic edits into the genome of a microbial host cell in an iterative fashion that does not require the use of functional counterselection following at least one round of transformation. It can be used to rapidly stack genetic edits in the genome of a microbial host cell. Kits for performing the methods are also disclosed.