The present disclosure provides compositions and methods for template-free double stranded geometric enzymatic nucleic acid synthesis of arbitrarily programmed nucleic acid sequences.

A method for constructing a gene mutation library, also relating to the gene mutation library obtained by the method, a kit for the method for constructing a gene mutation library, and a method for analyzing the relationships between amino acid mutations in a protein and the properties, regulation, and/or function of the protein using the gene mutation library constructed by the method.

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

A series of hybridisations is performed for forming a target double-stranded nucleic acid from initial fragments, where each further hybridisation step hybridises the direct products of a pair of earlier hybridisation steps. For at least one further hybridisation step H.sub.F, both of the corresponding pair of earlier hybridisation steps H.sub.E comprise an error-detecting type of hybridisation step, which includes an error detecting operation to detect whether the hybridised fragments formed in the error-detecting type of hybridisation step H.sub.E comprise at least one erroneous hybridised fragment, and discarding at least part of the erroneous fragment to exclude it from a subsequent further hybridisation step. By detecting and removing erroneous fragments throughout a staged and controlled hybridisation process, erroneous fragments are prevented from diluting the pool of error-free fragments at each hybridisation step, to improve yield.

Described herein is a Mycobacterium mutant, comprising at least one mutation in at least one gene sequence encoding global gene regulators (GGRs) selected from the group consisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein the GGR gene is at least partially inactivated. Described herein also is a vaccine based on the mutant and a method of differentiating between subjects that have been infected with Mycobacterium and subjects that have not been infected with Mycobacterium or have been vaccinated with a Mycobacterium vaccine.

A system for DNA gene assembly that utilizes a DNA symbol library and a DNA linker library. The symbol library has a number of DNA symbols each having a first overhanging end and a second overhanging end different than and non-complimentary to the first end, the first and second ends being the same nucleotides for each DNA symbol. The linker library has pairs of DNA linkers, a first linker of a pair having a first end and a second end and a second linker of the pair having a first end and a second end, the first end of the first linker being the same nucleotides for each first linker and the second end of the second linker being the same nucleotides for each second linker, wherein the second end of the first linker and the first end of the second linker have complementary nucleotides. The first linker joins to the first end of a DNA symbol and the second linker joins to the second end of another DNA symbol.

The invention provides methods for synthesizing a product DNA molecule of any possible DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence. In any embodiment the product DNA molecule has an error rate of less than 1 error per 2,000 nucleotides.

The present invention provides to an improved high-throughput system and method for generated and screening of genetic variants by combinatorial modifications. Also provided are optimized SpCas9 enzyme variants produced by this system.

The present invention provides to an improved high-throughput system and method for generated and screening of genetic variants by combinatorial modifications. Also provided are optimized SpCas9 enzyme variants produced by this system.

Methods and compositions relate to the assembly of high fidelity nucleic acids. Specifically, nucleic acid molecules having a desired predetermined sequence can be assembled after failure to assemble in conventional assembly. Aspects of the disclosure relate to methods of assembling a target nucleic acid molecule having a desired or predetermined sequence.