Biology subjects

ma, l.

Publications and source records attributed to ma, l..

4 recordsLinked to original sources

bioRxiv2022

Rational design of Cas9 ribonucleoprotein with a "gRNA-shRNA" for multidimensional genome manipulation and enhanced homology-directed repair

Gene perturbation approaches have evolved as powerful tools for understanding the function of genes and curing inherited diseases. Here, we develop a method that combines the merits of RNAi and CRISPR technology by rational design of Cas9 ribonucleoprotein (RNP) with a "gRNA-shRNA" component. The RNP, termed Cas9-RNAi, has a gRNA containing a 3 extension that can be processed to a functional siRNA via dorsha/dicer enzyme mediated cleavage within cells. We prepared the Cas9-RNAi RNPs by streamline co-expression of Cas9 enzymes and the "gRNA-shRNA" ribonucleotides in Escherichia coli strain HT115(DE)3. Transferring the Cas9-RNAi RNPs into mammalian cells achieves multidimensional genome manipulation, e.g., simultaneously knock out and knock down the target genes. Moreover, by introduction of a shRNA against the gene of human DNA ligase 4 (LIG4), significantly improved homology-directed repair was attained. Together, we develop a simple-to-use CRISPR RNP tool that has great potentials in precise genome editing, gene function analysis and gene therapy.

bioengineering↗

bioRxiv2019

Genome-wide developed microsatellites reveal a weak population differentiation in the hoverfly Eupeodes corollae (Diptera: Syrphidae) across China

The hoverfly, Eupeodes corollae, is a worldwide natural enemy of aphids and a plant pollinator. To provide insights into the biology of this species, we examined its population genetic structure by obtaining 1.15-GB random genomic sequences using next-generation sequencing and developing genome-wide microsatellite markers. A total of 79,138 microsatellite loci were initially isolated from the genomic sequences; after strict selection and further testing of 40 primer pairs in eight individuals, 24 polymorphic microsatellites with high amplification rates were developed. These microsatellites were used to examine the population genetic structure of 96 individuals from four field populations collected across southern to northern China. The number of alleles per locus ranged from 5 to 13 with an average of 8.75; the observed and expected heterozygosity varied from 0.235 to 0.768 and from 0.333 to 0.785, respectively. Population genetic structure analysis showed weak genetic differentiation among the four geographical populations of E. corollae, suggesting a high rate of gene flow reflecting likely widespread migration of E. corollae in China.

molecular biology↗

bioRxiv2018

Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step

CRISPR/Cas ribonucleoprotein (RNP) complexes have been recently used as promising biological tools with plenty of applications, however, there are by far no efficient methods to prepare them at large scale and low cost. Here, we present a simple method to directly produce and purify Cas RNP, including the widely used Cas9 and Cas12a nuclease, from E.coli in a single step using an ultra-high-affinity CL7/Im7 purification system. The prepared Cas RNP shows high stability, solid nuclease activity in vitro, and profound genome editing efficiency in vivo. Our method is convenient, cost-effective, and applicable to prepare other CRISPR associated nucleases.

synthetic biology↗

bioRxiv2018

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

synthetic biology↗