CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering

Abstract

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability

“…This resulted in a list of 81 genes with highly heterogenous molecular functions (Figure 1B, Supplemental Table 1) predicted to produce LOF models with protein-coding insertions and deletions (indels) confirmed via Synthego ICE analysis (https://ice.synthego.com). Given preliminary >90% cutting efficiency, a shotgun gene targeting approach[20], and a gene list highly intolerant to LOF, any transcript that failed to transmit (>8 pools of 5 F 1 larvae) to F 1 germline was assumed likely embryonic lethal. Additionally, F 1 fish that did not result in viable heterozygous (HET) in-cross (after >10 breeding opportunities) and thus did not provide F 2 offspring for downstream analysis were not pursued further.…”

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability

“…A single mutation (two if the zebrafish gene is duplication) was chosen per gene that was predicted to produce an LOF insertion or deletion (indel) in a protein-coding region, as confirmed by Synthego ICE analysis. Given preliminary >90% cutting efficiency, a shotgun gene targeting approach, 20 and a gene list highly intolerant to LOF, any transcript that failed to transmit (>8 pools of 5 F 1 larvae) to F 1 germline was assumed likely embryonic lethal. Additionally, F 1 fish that did not result in viable heterozygous (HET) in-cross (after >10 breeding opportunities) and thus did not provide F 2 offspring for downstream analysis were not pursued further.…”

Engineering yeast for the production of plant terpenoids using synthetic biology approaches

“…67 The application of COMPASS to the production pathway of b-ionone led to a 4.2-fold increase over the control strain. 67 CRISPR-Cas9 system has been used to make untargeted genome-wide modications 70 to randomly shi the gene expression across the genome to improve the production of a target product. The CRISPR-Cas9 assisted random mutation (CARM) technique was developed to introduce non-oriented genomic modications into the yeast genome using a random gRNA library 70 until acquiring the improvement of a target compound (Fig.…”

“…Thus, several rounds of CARM mutagenesis in the BY4741 yeast strain resulted in a 10.5-fold improvement in b-carotene production via altering the expression level of over 2500 distinct genes. 70…”