The invention concerns fusion proteins, wherein two endoglycosidases are fused, possibly via a linker. The fusion enzymes according to the invention have structure (1): EndoX-(L).sub.p-EndoY (1), wherein EndoX is an endoglycosidase, EndoY is an endoglycosidase distinct from EndoX, L is a linker and p is 0 or 1. Such fusion enzymes capable of trimming glycoproteins comprising at least two distinct glycoforms in a single step. The invention further concerns the use of the fusion enzyme according to the invention for trimming glycoproteins. In another aspect, the invention relates to the process of production of the fusion enzyme. In a further aspect, the inventions concerns a process for trimming glycoproteins, comprising trimming the glycoprotein with a fusion enzyme according to the invention, to obtain a trimmed glycoprotein.

The invention concerns fusion proteins, wherein two endoglycosidases are fused, possibly via a linker. The fusion enzymes according to the invention have structure (1): EndoX-(L).sub.p-EndoY (1), wherein EndoX is an endoglycosidase, EndoY is an endoglycosidase distinct from EndoX, L is a linker and p is 0 or 1. Such fusion enzymes capable of trimming glycoproteins comprising at least two distinct glycoforms in a single step. The invention further concerns the use of the fusion enzyme according to the invention for trimming glycoproteins. In another aspect, the invention relates to the process of production of the fusion enzyme. In a further aspect, the inventions concerns a process for trimming glycoproteins, comprising trimming the glycoprotein with a fusion enzyme according to the invention, to obtain a trimmed glycoprotein.

A Streptococcus canis Cas9 (ScCas9) ortholog and its engineered variants, possessing novel PAM specificity, is an addition to the family of CRISPR-Cas9 systems. ScCas9 endonuclease is used in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. A novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii and Streptococcus angionosis facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

A Streptococcus canis Cas9 (ScCas9) ortholog and its engineered variants, possessing novel PAM specificity, is an addition to the family of CRISPR-Cas9 systems. ScCas9 endonuclease is used in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. A novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii and Streptococcus angionosis facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.

Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS) and for expressing proteins in cells. Particularly disclosed are vectors comprising Golden Gate sites for cloning, methods for preparing such vectors, and the use thereof for performing CFPS and for expressing proteins in cells such as in naturally occurring or recombinant species of Clostridia, including Clostridium autoethanogenum.

Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS) and for expressing proteins in cells. Particularly disclosed are vectors comprising Golden Gate sites for cloning, methods for preparing such vectors, and the use thereof for performing CFPS and for expressing proteins in cells such as in naturally occurring or recombinant species of Clostridia, including Clostridium autoethanogenum.