Application Note
OligoMix® for Production of Cloned Libraries
The use of synthetic oligonucleotides for cloning complex libraries of constructs with predefined nucleic acid sequences is now a common practice. Because the synthesis and cloning steps are expensive and individual manipulation is labor intensive, researchers have adopted the use of oligonucleotide pools over individually synthesized oligos.
OligoMix® enables us to synthesize thousands of designed sequences at once on a single microarray chip. By synthesizing the sequences in massive-parallel on a microfluidic chip, the overall cost and time required for synthesis is dramatically decreased and the cumbersome procedure of multiple transformation reactions for all unique constructs is avoided.
Production of cloned libraries with microarray-based oligo synthesis provides a rapid, high-throughput, cost-effective approach for the generation of complex libraries of designed oligo sequences. The flexibility to create fully customized sequences means this approach can address an array of biological questions. Applications include: short hairpin RNA (shRNA) libraries for high-throughput loss-of-function genetic screens and antibody or other protein-coding DNA libraries for diversity studies and directed evolution strategies1.
OligoMix® as CRISPR Guide RNAs
Recently, a new application of oligonucleotide libraries has emerged. The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a “game changing” technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications, as well as for large-scale functional genomics.
CRISPRs are segments of prokaryotic DNA containing short repetitions of base sequences. In recent years, it’s been discovered that a set of genes (known as cas, or CRISPR associated, genes) encode helicase or nuclease proteins that induce site-directed double strand breaks in these repetitive regions of DNA. Through non-homologous end joining and homologous recombination these breaks are repaired to produce targeted mutations (edits). Of particular interest to molecular biologists is the Cas9 protein which utilizes guide RNAs (gRNAs) to interrogate and cleave invading DNAs which are complimentary to the guide RNA’s 20 basepair spacer region. By modifying this spacer region on the Cas9 guide RNA, the Cas9 protein can introduce site-directed double strand breaks in DNA to inactivate or edit specific genes of interest.
In CRISPR-Cas9 mutation screens, guide RNAs targeting tens of thousands of sites within genes are cloned into viral vectors and delivered as a pool into target cells along with Cas9. By identifying guide RNAs that are enriched or depleted in cells which exhibit a desired phenotype, researchers can systematically identify genes that are required for that particular phenotype. By scaling the guide RNA synthesis processes to massively parallel arrays, screening assays at unprecedented throughput are made possible. LC Sciences’ OligoMix® offers a unique solution for researchers looking to generate large guide RNA libraries, as users are able create fully designed libraries of thousands of specific, single-stranded oligonucleotide sequences for recognizing particular genomic regions.
CRISPR/Cas9 Single Guide (sg)RNA Libraries in All-in-one Retroviral Vectors
Researchers at McGill University demonstrate that cloning oligonucleotide pools into retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs, provides stable and reproducible expression of the editing tools for high-throughput functional genomic screens2.
Oligonucleotides are individually synthesized en masse on a microarray chip. These are then PCR amplified to incorporate vector compatible restriction sites. Library pools of guides are then used for screening purposes. Following isolation of genomic DNA from positively selected cells, amplification by PCR across the guide region is performed and the guide identified by sequencing. Modification at the expected locus is then confirmed using the T7 endonuclease I assay, SURVEYOR assay, or sequencing of PCR products.
Schematic representation of sgRNA library generation and pooled screening strategy
CRISPR /Cas9-mediated Functional Screening of cis-Regulatory Elements
Recently, researchers from the Ludwig Institute for Cancer Research developed a high-throughput CRISPR/Cas9-based genome-editing strategy for functional screening of cis-regulatory elements in the human genome3. Application of this strategy to POU5F1 in the human embryonic stem cells (hESCs) uncovered both classical cis-regulatory elements and a class of noncanonical elements that regulate transcription in an unexpected manner.
Single guide RNA library sequences were designed to create random mutations at 174 predicted regulatory regions in the POU5F1 TAD via nonhomologous end joining (NHEJ). Single guide RNA sequences were synthesized in an array-based oligo pool (OligoMix®), cloned into the lentiCRISPR plasmid, and packaged into lentiviral libraries. The researchers then used lentiviral libraries to infect H1 POU5F1-eGFP cells to generate random mutagenesis of the 174 candidate regions. hESCs with mutations at cis-regulatory elements affecting POU5F1 expression can be identified as eGFP− cells. The eGFP− P4 population was collected by FACS sorting. Genomic DNA was isolated from P4 and nonsorted control cells, followed by PCR amplification of single guide RNA sequence and deep sequencing.
Experimental design of a high-throughput CRISPR/Cas9-mediated screen
for identifying cis-regulatory elements
(A) Workflow of lentiCRISPR screening strategy to identify functional regulatory elements. (B) Control H1 POU5F1-eGFP without lentiviral infection were dissociated into single cells and subjected to FACS analysis to determine the eGFP− (P4) gate for eGFP− population; 0.31% indicates the ratio of P4 in parental live singlets. (C,D) The H1 POU5F1-eGFP cells were infected with lentiCRISPR library by spin infection at low multiplicity of infection (MOI). Twenty-four hours after infection, the cells were cultured for 7 d under puromycin selection; for another 10 d, without puromycin. (C) The cells were subjected to FACS analyses. (2.16%) The ratio of P4 in parental population. (D) Scatter plot for sgRNA read counts in eGFP− cells compared with the control cells after LOESS normalization. Dots underneath the green line are sgRNAs with at least twofold enrichment in the eGFP− cells compared with the control population.
The researchers performed the experiment four times and identified a list of single guide RNA sequences with at least twofold enrichment in the eGFP− population in each experiment. These elements are located with various linear genomic distances from the POU5F1 transcription start site (TSS), ranging from −1.4 to 491 kbp. Among these positive hits are the POU5F1 promoter and a proximal enhancer (DHS_115) located 1.4 kbp upstream of the TSS, which confirms the essential role of POU5F1 promoter in controlling gene expression, and also provides additional functional evidence for the POU5F1 proximal enhancer.
These results demonstrate the utility of high-throughput screening for functional characterization of noncoding DNA and reveal a previously unrecognized layer of gene regulation in human cells.
High-throughput Mapping of Regulatory DNA
Researchers from MIT and Brigham and Women’s Hospital recently showed that OligoMix® could be used to generate guide RNA libraries and demonstrated their effectiveness in an elegant CRISPR-Cas9–based multiplexed editing regulatory assay (MERA) screening method4.
In MERA, a genomically integrated dummy guide RNA is replaced with a pooled library of guide RNAs through CRISPR-Cas9–based homologous recombination such that each cell receives a single guide RNA. Guide RNAs are tiled across the cis-regulatory regions of a GFP-tagged gene locus, and cells are flow cytometrically sorted according to their GFP expression levels. Deep sequencing on each population is used to identify guide RNAs preferentially associated with partial or complete loss of gene expression.
Multiplexed editing regulatory assay (MERA)
(a) MERA workflow. (b) Zfp42GFP mESCs show uniformly strong GFP expression. After bulk gRNA integration, a subpopulation of cells lose GFP expression partially or completely. These cells are flow cytometrically isolated for deep sequencing. (c,d) Bulk reads for gRNAs are highly correlated between replicates from the Tdgf1 (c) or Zfp42 libraries (d), indicating consistent and replicable integration rates.
The team’s multiplexed editing regulatory assay (MERA) utilized non-homologous end-joining, induced by CRISPR-Cas9, to produce a range of indels in the GFP-tagged locus of four mESC-specific, knock-in genes (Nanog, Rpp25, Tdgf1 and Zfp42 ). By designing four, respective guide RNA libraries to tile the cis-regulatory regions of each gene (each with 3,908 guide RNAs) the team was able to map elements required for gene expression and perform deep sequencing of the guide RNA-induced mutations to reveal genotypes that either did or did not lose gene expression. The results of their MERA screen were validated through replacement of selected genomic elements by homologous recombination.
These results demonstrate that OligoMix® can be used effectively in the design of guide RNA libraries for use in CRISPR Cas 9 mutation screens. Using these tools to study genes involved with survival, drug resistance and tumor metastasis could allow researchers to pioneer new disease treatment discoveries.
References
- Xu M, Hu S, Ding B, Fei C, Wan W, Hu D, Du R, Zhou X, Hong J, Liu H. (2015) Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides. Journal of Biotech 194(1), 27-36. [article]
- Malina A, Katigbak A, Cencic R, Maïga RI, Robert F, Miura H, Pelletier J. (2014) Adapting CRISPR/Cas9 for functional genomics screens. Methods Enzymol 546:193-213. [article]
- Diao Y, Li B, Meng Z, Jung I, Lee AY, Dixon J, Maliskova L, Guan KL, Shen Y, Ren B. (2016) A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res 26(3):397-405. [article]
- Rajagopal N, Srinivasan S, Kooshesh K, Guo Y, Edwards MD, Banerjee B, Syed T, Emons BJ, Gifford DK, Sherwood RI (2016) High throughput mapping of regulatory DNA. Nat Biotechnol 34(2):167-74. [article]