Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments

Designing a Framework for Investigating the Role of Active Transcription, Mismatch Repair and Chromatin Accessibility in Prime Editing

Although CRISPR/Cas systems have revolutionized gene editing, their applications in clinical therapies and research are still limited by their reliance on double-strand breaks (DSBs). Prime editing (PE) is a new technology which utilizes a Cas9 nickase fused to a reverse transcriptase to directly transcribe small, precise edits at target sites, foregoing the need for DSBs. Although this technique is accurate in introducing small edits into the genome, it is still variable in its editing efficiency and a better understanding of the factors which may control these PE efficiencies is needed. In this project, I first tested the role of active transcription around the edit site, finding that modulating transcription around a reporter edit site did not impact edit efficiency of PE. As mismatch repair (MMR) had previously been implicated in affecting PE, I knocked down the MMR gene MSH3 and designed pegRNAs to find edits sensitive to this knockdown. Finally, I attempted and failed to replicate CRISPRoff silencing of eGFP close to a PE target site in order to investigate the role of chromatin accessibility on PE. However, I did formulate a new design for CRISPRoff silencing that could do this reliably. While this study did not find conclusive results on the effects of active transcription, MSH3 and chromatin accessibility on PE, it did provide experimental frameworks for future studies that may do this. This future research will better inform the cellular determinants of PE efficiencies, allowing PE to be further optimized for therapeutic and research applications

Changes in Enzyme Activities in Salt-Affected Soils during Incubation Study of Diverse Particle Sizes of Rice Straw

Soil enzymes are linked to the plant–soil–enzyme–soil nutrients of the soil system, which play an important role in carbon cycling and phosphorus mineralization in soil. Monitoring soil biological quality, particularly enzyme activities, after receiving organic amendments is a prerequisite for the sustainable management of soils. An incubation study was conducted to evaluate the effect of different particle sizes of rice residue (control, powdered, 1 cm, 2 cm, 5 cm, and 10 cm) on the enzymatic activities in three soils (normal, saline, and sodic). The soils used in the study were alkaline in reaction with a pH range of 7.05–8.86 and an electrical conductivity (EC) gradient from 0.41 to 2.5 dS m−1. Significant changes in the soil enzyme activity (dehydrogenase, fluorescein diacetate, and alkaline phosphatase) were observed with the incorporation of rice residue as compared to control. The enzymatic activities were substantially enhanced with a decrease in the size of the residue up to 28 days during the incubation period. The maximum enzymatic activity in the three soils was found to be in the order of normal > sodic > saline soils. These results suggest that the particle size of rice residues and salt levels should be considered important factors in residue decomposition in soils, as they directly influence the activity of soil enzymes for the overall improvement of the biological pools in soils

Table2_Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.docx

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.</p

Table1_Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.docx

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.</p