Optimizing in vitro Transcribed CRISPR-Cas9 single-guide RNA Libraries for Improved Uniformity and Affordability

This method describes a scalable sgRNA synthesis workflow that reduces costs by over 70% by combining large pools of microarray-derived oligos encoding unique sgRNA spacers. Sub-pool oligos are converted into full-length templates through Golden Gate assembly before in vitro transcription by T7 RNA polymerase. RNA-seq revealed dramatic skewing in spacer representation within libraries, with some spacers dominating while others dropped out entirely. We identified guanines within the first four nucleotides of the spacer sequence immediately downstream of the T7 promoter as the primary driver of this bias. The addition of a guanine tetramer in front of all spacers reduced bias by a mean of 19% in sgRNA libraries containing 389 spacers, but increased the amount of unwanted high-molecular weight RNA species present after transcription. Two alternative strategies to mitigate bias were also tested: compartmentalizing spacers within emulsions and varying DNA input and reaction volumes. Both methods independently reduced bias in sgRNA libraries containing 2,626 spacers. These advancements improve the affordability and quality of sgRNA libraries, with broad implications for enhancing CRISPR-Cas9 screens and guide RNA library design for other CRISPR or nuclease systems.