Project description:The use of CRISPR/Cas proteins for the creation of multiplex genome-engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR/Cas9 system for multiplex genome-engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome-editing in Arabidopsis thaliana. We identified optimized promoter sequences for driving expression of single transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome-engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome-editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex-genome-engineering in plant-based systems.
Project description:we characterized a novel compact Cas12a ortholog, EbCas12a, from the Erysipelotrichia bacterium with activities in mammalian cells. It is with the PAM sequence of 5’-TTTV-3’ (V=A, G, C) and the smallest size of ~3.47kb among reported Cas12a orthologs so far. Moreover, enhanced EbCas12a (enEbCas12a) was also developed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells. With the help of the compact enEbCas12a, all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo. Altogether, with the help of the novel smallest high fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
2021-09-10 | GSE180773 | GEO
Project description:NGS of crRNA arrays used for multiplex pathway engineering
Project description:CRISPR/Cas12a-based combinational screening has shown remarkable potential in identifying genetic interactions. Here, we described an innovative method for combinational genetic screen with rapid constructing of dual-crRNA library by gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which was identified previously by us with strict PAM recognition and low off- targeting, to guarantee the fidelity and efficiency. The custom, pooled SOE crRNA array (SOCA) library for double knockout screen could be conveniently constructed in lab for widespread use and CeCas12a mediated high fidelity screen display good performances even under negative selection screen. By designing an SOCA dual-crRNA library which covered the most of kinase and metabolism-associated gene targets of FDA- approved drugs that were implicated in hepatocellular carcinoma (HCC) tumorigenesis, novel cross talks between the two gene sets were negatively selected out to synergistically inhibit HCC cell growth in vitro and in vivo and also validated by virtual double knockdown screening based on TCGA databases. Thus through our rapid, efficient and high fidelity double knockout screening system, it is very promising to systemically dig genetic interactions between multiple gene sets or synergistic combinations of FDA-approved drugs for clinical translational medicine in the future.
Project description:CRISPR/Cas12a-based combinational screening has shown remarkable potential in identifying genetic interactions. Here, we described an innovative method for combinational genetic screen with rapid constructing of dual-crRNA library by gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which was identified previously by us with strict PAM recognition and low off- targeting, to guarantee the fidelity and efficiency. The custom, pooled SOE crRNA array (SOCA) library for double knockout screen could be conveniently constructed in lab for widespread use and CeCas12a mediated high fidelity screen display good performances even under negative selection screen. By designing an SOCA dual-crRNA library which covered the most of kinase and metabolism-associated gene targets of FDA- approved drugs that were implicated in hepatocellular carcinoma (HCC) tumorigenesis, novel cross talks between the two gene sets were negatively selected out to synergistically inhibit HCC cell growth in vitro and in vivo and also validated by virtual double knockdown screening based on TCGA databases. Thus through our rapid, efficient and high fidelity double knockout screening system, it is very promising to systemically dig genetic interactions between multiple gene sets or synergistic combinations of FDA-approved drugs for clinical translational medicine in the future.