SUNi mutagenesis: Scalable and uniform nicking for efficient generation of variant libraries

Abstract

Multiplexed assays of variant effects (MAVEs) have made possible the functional assessment of all possible mutations to genes and regulatory sequences. A core pillar of the approach is generation of variant libraries, but current methods are either difficult to scale or not uniform enough to enable MAVEs at the scale of gene families or beyond. We present an improved method called Scalable and Uniform Nicking (SUNi) mutagenesis that combines massive scalability with high uniformity to enable cost-effective MAVEs of gene families and eventually genomes.

Fig 1. Optimization and analysis of nicking mutagenesis primer design.

a, Per position mutation frequency presented as fraction of all sequencing reads for standard nicking. Dashed lines indicate 90^th and 10^th percentile of all mutation frequencies. b, Per position mutation frequency presented as fraction of all sequencing reads for opt1 nicking. Dashed lines indicate 90^th and 10^th percentile of all mutation frequencies. c, Spearman correlation between GC content of the 5’ terminus and mutagenesis efficiency, when considering between one and five terminal bases. d, Mutagenesis frequency of positions with different GC content in the 5’ terminal three bases. Spearman ρ = 0.56, p = 6.8x10^-8. e, Mutagenesis frequency of positions with different SW sequences (S = G or C, W = A or T) in the 5’ terminal three bases.

Fig 2. Performance and comparison of SUNi mutagenesis with other methods.

a, Per position mutation frequency presented as fraction of all sequencing reads for SUNi mutagenesis. Dashed lines indicate 90^th and 10^th percentile of all mutation frequencies. b, Screening efficiency of different mutagenesis methods. c, Screening efficiency of different mutagenesis methods, as a function of uniformity and percent programmed. Colors the same as in b.