Assembly of all sequence information revealed that the vector sequence had been re-organized prior to integration as shown in Fig. All three mutants contained the same integrated sequence, suggesting that they arose from a single transformation event.
We also characterized their integration in the genome by performing the Southern blot, and found that the integration was a single copy insertion with the same size of restriction fragments Fig. Amplification of fragments encompassing the cut site failed under a short elongation cycle upper panel , but improved when we used a longer cycle lower panel.
The specific primers used in this study are listed in Table S1. The guide sequence was searched on the Chlamydomonas reference genome sequence allowing 3 mismatches and 2 bulges, and a total of off-target candidate sites could be identified.
Among these, we were able to locate sites on our WT CC genomic sequence that matched those of the Chlamydomonas reference sequence CC 4. We surveyed these sequences on the genomic sequences of CpSRP43 , and , and confirmed the presence of sites. Vector pCr containing the hygromycin resistance marker was co-transformed for selection purpose. We obtained about initial transformants. From them, we isolated 10 clones that showed yellow or brown colony color, and designated ChlM-3, -4, -9, , , , , , , Fig.
Visual assessment of coloration Fig. Southern blot analyses were performed to characterize genomic integration patterns after digestion of their genomic DNAs with Nco I that does not cut the vector Fig.
Most of the selected transformants contained a single copy of the vector except for ChlM-4 that contained two copies.
Two pairs of clones ChlM and ChlM; ChlM and ChlM showed the same banding patterns, suggesting that they were duplicate clones representing the same integration events. We thus observed at least eight independent integration events, most of which were single-copy integration of the vector. IDA5 was detected as a loading control. We tested combinations of primers Table S1 located in the ChlM locus and the vector, and successfully determined the junction sequences for six clones including ChlM-3, -9, , , and Fig.
All of them showed integration of the vector at the Cas9 cut site, and were characterized by different rearrangements of vector sequences and different small indels next to the Cas9 cut site. ChlM and ChlM showed the same vector sequence, but contained different deletions at the junctions Fig. Our results indicate that the 10 isolated mutants represent at least nine independent integration events with extensive rearrangements of the vector sequences.
Interestingly, the six clones with identified junction sequences were examples of NHEJ-mediated knock-in of the vector, because the vector contained no sequences homologous to the ChlM locus.
We failed to identify the integration junction sequences for the remaining four clones; however, ChlM-4, ChlM and ChlM showed the same bands hybridized to both probes of ChlM and aphVII , suggesting that they are also knock-ins of the vector sequence. It should be noted that ChlM-4 contained two copies of vector sequences: one at the ChlM locus and the other at an unknown location, based on the Southern blot Fig. These mutations could be categorized into three classes as summarized in Fig.
S5e : simple knock-ins ChlM-3, -9, , , , , , , knock-in plus integration at another location ChlM-4 , and deletion plus integration at other location ChlM A Multiple PCR fragments encompassing the vector sequences. B — G Schematic molecular maps of the knock-in events in each ChlM mutant. The sizes of the inserted vector and PCR amplification are shown.
Red and black letters indicated the deleted and inserted sequences, respectively. This improvement may be attributed to the delivery of the Cas9 RNP, as employed successfully in other organisms 28 , 30 , Vector-driven expression of Cas9 may be toxic to Chlamydomonas , probably due to the continuous expression Delivery of purified Cas9 RNP may have other advantages including technical simplicity and fewer occurrences of off-target events 30 , Occurrence of these targeted mutations was highest when cells were delivered with the highest amount of Cas9 and MAA7 -2 sgRNA Table 1 , even though we have not determined optimum conditions that might have to be determined empirically.
The error-prone double strand break DSB repair of DNA must be involved in generation of small indel mutations 46 , and the mechanism of DSB repair in Chlamydomonas might have played a role in the unique pattern of mutations obtained in our mutagenic screen.
Alternatively, complete loss-of-function mutations could not survive the selection conditions employed in our auxotrophic screens.
Even though tryptophan was supplemented in the media, uptake of tryptophan in Chlamydomonas is not known, since no true loss-of-function mutations of TSB have been characterized 17 , It is interesting to note that the integration of the vector sequences at the Cas9 cut sites of CpSRP43 and ChlM was mediated by NHEJ, since the co-transformed vectors did not contain any sequence from the target genes or the neighboring loci. Knock-in can be achieved by homology-driven recombination HDR wherein the vector sequence contains wings of target-flanking sequences, or by NHEJ that does not require any cloning of the wings 24 , 47 , 48 , 49 , 50 , It is not clear why we obtained predominantly knock-in events.
It is possible that DSBs induced by Cas9 facilitated integration of available free ends of co-transformed vectors at the cut site.
Our present finding opens up a range of possible applications of NHEJ-mediated knock-in, including insertion of a gene at a specific location without requiring the cloning of homologous sequences. The direct delivery of Cas9 RNPs may have other advantages including minimized off-targeting events, less toxicity of Cas9, and no laborious cloning work compared to the transgenic techniques In our mutagenic screen targeting the CpSRP43 locus, sequencing the whole genomes of cpsrp43 mutants revealed no incidences of off-targeting by using Cas-OFFinder We also found predominant NHEJ-mediated knock-in events when co-transformed with unrelated vectors used for the selection purpose.
This work can be applied not only to biological research in Chlamydomonas , but also to industrial applications of genome editing in other microalgae for large scale production of biofuels and other biomaterials. We chose target genes based on our ability to identify mutations via auxotrophic or visual means.
Mutations in the other two tested genes could be selected based on the lighter colony color arising from reduced chlorophyll synthesis 34 , For the latter two genes, we co-transformed cells with a hygromycin-resistance vector and used antibiotic selection to improve the delivery efficiency in Chlamydomonas. Their locations along each locus are shown in Fig. All strains were maintained on Tris-acetate-phosphate TAP agar plates [2. The activities of the sgRNAs and the Cas9 protein were verified in vitro prior to their use in vivo.
Briefly, cells were harvested by centrifugation and genomic DNAs were extracted according to the instructions provided with the Instagene Matrix. The final products were separated by 0. For transformation of C. Mutant cells were selected on TAP agar plates containing 1. After recovery, transformed cells were spread on TAP agar plates containing 1. Chlorophyll contents were measured using methanol extraction according to published protocols The concentrations of chlorophyll a and b were calculated as follow:.
Genomic DNA was extracted according to published methods 58 , The SolexaQA package software version 1. The trimmed reads were assembled using the SOAPdenovo version 2. The assembled contigs were aligned with the C. For the alignment parameters, the gap-open cost and gap-extension cost were set at A phylogenetic tree of the aligned sequences was constructed using the neighbor-joining method, and the Jukes-Cantor model was used to measure protein distance.
The cell pellets were resuspended with 1. The primers used to generate each probe are listed in Table S1. How to cite this article : Shin, S. Ho, S. Perspectives on engineering strategies for improving biofuel production from microalgae - A critical review. Biotechnol Adv 32 , — Wijffels, R. An outlook on microalgal biofuels. Science , — Harris, E. Chlamydomonas as a model organism.
Merchant, S. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Mussgnug, J. Genetic tools and techniques for Chlamydomonas reinhardtii. Kang, N. Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina. Biotechnol Biofuels 8 , Article Google Scholar. Yao, Y. Glycerol and neutral lipid production in the oleaginous marine diatom Phaeodactylum tricornutum promoted by overexpression of glycerolphosphate dehydrogenase.
Select personalised ads. Apply market research to generate audience insights. Measure content performance. Develop and improve products. List of Partners vendors. A knock-in option is a latent option contract that begins to function as a normal option "knocks in" only once a certain price level is reached before expiration.
Knock-ins are a type of barrier option that are classified as either a down-and-in or an up-and-in. A barrier option is a type of contract in which the payoff depends on the underlying security's price and whether it hits a certain price within a specified period.
Knock-in options are one of the two main types of barrier options, with the other type being knock-out options. A knock-in option is a type of contract that is not an option until a certain price is met. So if the price is never reached, it is as if the contract never existed.
However, if the underlying asset reaches a specified barrier, the knock-in option comes into existence. The difference between a knock-in and knock-out option is that a knock-in option comes into existence only when the underlying security reaches a barrier, while a knock-out option ceases to exist when the underlying security reaches a barrier.
Barrier options typically have cheaper premiums than traditional vanilla options , primarily because the barrier increases the chances of the option expiring worthless. Gene knockout and gene knockdown are two mechanisms of silencing the expression of genes inside organisms. Gene knockout is a laboratory technique of gene silencing responsible for the complete erasing of the gene from the genome or the inactivation of the gene through nonsense mutations by the introduction of frameshift mutations or stop codons to the gene sequence.
As the blueprint of the gene is destroyed, the target gene product is also ablated. The method was originally developed with homologous recombination. It involves the delivery of a DNA construct, which contains the desired mutation.
Then, this construct is recombined with the target gene, completely removing the gene sequence from the genome. A regulator region of DNA a short distance from the 5' end of a gene that acts as the binding site for RNA polymerase. A sequence of DNA that is designed with at least 1 a splice acceptor to insert itself into genes and 2 a selection cassette to disrupt transcription.
Polymerase chain reaction- a method for amplifying specific DNA segments which exploits certain features of DNA replication. Transfer of electrophorectically separated fragments of DNA from the gel to an absorbent sheet such as paper. This sheet is then immersed in a solution containing a labeled probe that will bind to a fragment of interest. Skip to content. Anderson Angela M. Belcher Sangeeta N. Cima Paula T. Housman Richard O. Hynes Darrell J. Langer Jacqueline A.
Lees J. White K. Dane Wittrup Michael B. Preclinical Modeling Facility. Knockins and Knockouts The ability to engineer the mouse genome has proven useful for a variety of applications in research, medicine and biotechnology.
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