A method for scarless genome editing is disclosed. In particular, the method provides scarless genome modification by using homology directed repair (HDR) steps to genetically modify cells and remove unwanted sequences. This method can be used for genome editing, including introducing mutations, deletions, or insertions at any position in the genome without leaving silent mutations, selection marker sequences, or other additional undesired sequences in the genome.

The invention encompasses compositions, kits and methods for modifying bacteria, preferably naturally occurring bacteria, in situ. These can be used to treat, prevent or cure microbiome-associated diseases or disorders by modulating the molecules expressed and/or secreted by bacterial populations of the microbiome in a specific manner. The genomic modifications can modify the interactions between part or all of these populations and the host in a way that decreases their deleterious potential on host health. The compositions, kits and methods of the invention do not result in the direct death of these populations or a direct significant inhibition of their growth. The invention further includes methods for screening for genetic modifications in the bacteria, for determining the efficiency of vectors at inducing these genetic mutations, and for determining the effects of these mutations on bacterial growth.

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

A transgenic chicken comprising an inactivated heavy immunoglobulin gene and/or inactivated light chain immunoglobulin gene is provided, as well as cells and targeting vectors for making the same.

A method for increasing the efficiency of homologous recombination-based gene editing in a plant according to an embodiment of the present invention includes optimizing temperature and photoperiod conditions during tissue culture of plant cells, expressing factors required for homology-directed DNA repair (HDR) and factors for increasing the HDR efficiency by using a multiple replicon, or regulating the HDR pathway or non-homologous end joining (NHEJ) pathway.

A cyanobacterium transformed using a gene transfer vector. The gene transfer vector includes: a first homologous recombination region homologous to a 5′ side of a target DNA of the cyanobacterium; a second homologous recombination region homologous to a 3′ side of the target DNA; and a DNA fragment introduced into a portion sandwiched between the first homologous recombination region and the second homologous recombination region. A total length of the first homologous recombination region and the second homologous recombination region is 5 kbp or more. The cyanobacterium has a DNA fragment transferred between the 5′ side of the target DNA and the 3′ side of the target DNA.

The present disclosure provides methods of generating recombinant bacteriophage genomes. Specifically, the present technology provides methods of integrating a heterologous nucleic acid sequence into a linear bacteriophage DNA genome, and isolating recombinant bacteriophages that express the heterologous nucleic acid sequence.

The present invention relates to a novel Chlamydomonas strain with an improved oil generation function, the strain of the present invention having useful mycological characteristics as a strain that provides a useful substance, such as a vegetable oil, in a microalga, as the strain has a fast cell growth speed and an excellent lipid generation function compared to conventional strains. In particular, the present invention can provide a vegetable oil with improved stability and a longer preservation period by containing, in a cell, a large amount of antioxidant pigments such as lutein and zeaxanthin, and can, thereby, be usefully used in industries such as food, medicine, cosmetics, etc., which utilize a vegetable oil.

A very fast and efficient CRISPR/Cas9 system is provided. Compositions include light-sensitive caged nucleotides at the PAM distal region of guide RNAs (gRNA.sup.caged) to create artificial mismatches as a “roadblock”. Upon light stimulation, the caging group (“roadblock”) is removed and the gRNA fully hybridizes with the target DNA. Thus, the pre-bound inactive Cas9/gRNA.sup.caged is rapidly converted to active Cas9.

The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides small Cas proteins and their use in modifying target sequences. In one aspect, the present disclosure provides a non-naturally occurring or engineered system comprising: a Cas protein that comprises a RuvC domain and a HNH domain, and is less than 850 amino acids in size; and a guide sequence capable of forming a complex with the Cas protein and directing the complex to bind to a target sequence.