CRISPR Toolkit

A curated collection of common CRISPR plasmids for genome editing research, including Cas9 systems, Cas12a systems, and base editors. This repository helps researchers quickly find reference sequences for widely used CRISPR approaches.

What’s in this repository

This collection includes many of the most cited and widely adopted CRISPR constructs from the Zhang, Liu, and Sabatini labs. All plasmids are sourced from Addgene and linked to their original publications.

Cas9 Systems (SpCas9)

pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) All-in-one SpCas9 vector with a guide RNA cloning site and puromycin selection for mammalian genome editing. From the Feng Zhang lab. Ran et al., Nat Protoc. 2013. doi: 10.1038/nprot.2013.143

pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene #42230) SpCas9 expression vector with a U6-driven guide RNA scaffold for efficient CRISPR editing in mammalian cells. Cong et al., Science. 2013. doi: 10.1126/science.1231143

lentiCRISPR v2 (Addgene #52961) Lentiviral delivery system for stable expression of Cas9 and guide RNA with puromycin selection. Sanjana et al., Nat Methods. 2014. doi: 10.1038/nmeth.3047

pSpCas9n(BB)-2A-Puro (PX462) V2.0 (Addgene #62987) Cas9 nickase (D10A) version designed to reduce off-target editing using paired guides. Ran et al., Nat Protoc. 2013. doi: 10.1038/nprot.2013.143

Cas12a (Cpf1) Systems

pY010-Cas12a (Addgene #74078) LbCas12a expression vector for mammalian genome editing with alternative PAM requirements. Zetsche et al., Cell. 2015. doi: 10.1016/j.cell.2015.09.038

pX551-Cas12a (Addgene #107633) Enhanced Cas12a variant engineered for improved activity in mammalian cells. Kleinstiver et al., Nat Biotechnol. 2019. doi: 10.1038/s41587-018-0011-0

Base Editors

pCMV-BE3 (Addgene #73021) Cytosine base editor enabling C→T or G→A transitions without introducing double-strand breaks. Komor et al., Nature. 2016. doi: 10.1038/nature17946

pCMV-ABEmax (Addgene #112095) Adenine base editor for A→G or T→C conversions with increased efficiency. Koblan et al., Nat Biotechnol. 2018. doi: 10.1038/nbt.4172

Looking for more base editors? See the dedicated base-editing-systems repository.

Delivery Systems

pLKO.1-puro-U6-sgRNA (Addgene #50920) Lentiviral guide RNA expression vector used alongside separate Cas9 delivery systems. Stewart et al., RNA. 2003. doi: 10.1261/rna.2530203

pAAV-CRISPR-SpCas9-sgExpression (Addgene #60958) AAV-compatible CRISPR vector for in vivo delivery of Cas9 and guide RNA. From Juan Jovel.

How to choose a CRISPR construct

This section summarizes typical use cases to help researchers pick the right vector.

For mammalian cell editing: pSpCas9(BB)-2A-Puro (PX459) V2.0 is one of the most widely used all-in-one CRISPR systems.

For stable genome editing: lentiCRISPR v2 enables long-term, integrated Cas9 expression.

For reducing off-target activity: Cas9 nickase (PX462) with paired guides or base editors (BE3, ABEmax) can help minimize unintended edits.

For targets without NGG PAM sites: Cas12a vectors offer alternative PAM recognition and may expand targetable regions.

For in vivo applications: AAV-compatible systems such as pAAV-CRISPR-SpCas9-sgExpression are optimized for animal studies.

Guide RNA design

Most plasmids use simple restriction-enzyme–based insertion (e.g., BbsI or BsaI sites). Researchers typically design 20 bp guide sequences using online tools such as:

• Benchling
• CRISPOR
• Synthego Guide Designer

Need custom CRISPR tools?

Visit Ambryon.com for:

• In vivo-ready plasmid and viral vectors
• Validated CRISPR tools tested in specific cell type/tissues
• All-in-one adeno-associated virus (AAV) gene editors
• Base-editor constructs with custom guides

All constructs are manufactured in the US with rigorous quality control and fast turnaround.

About these sequences

These plasmids are published, peer-reviewed CRISPR tools validated extensively in genome-editing research. All sequences originate from Addgene and cite the original publications and contributors.

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Questions? Email customer@ambryon.com or visit Ambryon.com to order manufacturing batches.