CRISPR proteins.<\/strong> Choose from several recombinant variants of Cas9 (Streptococcus pyogenes<\/em>), as well as Cas12a (formerly Cpf1<\/a>) (Acidaminococcus sp. BV3L6<\/em>) endonucleases. Alt-R S.p. Cas9 Nuclease V3 is suitable for most genome editing studies. Some experiments may benefit from use of Alt-R S.p. HiFi Cas9 Nuclease, a high-fidelity nuclease that has been engineered to reduce off-target effects<\/a> while retaining the on-target potency of wild-type Cas9. Alt-R Cas12a (Cpf1) nucleases provide additional genome editing target sites, because they recognize an AT-rich protospacer adjacent motif (PAM) rather than the CG-rich Cas9 PAM sequence. If you are interested in knock-in experiments or other applications requiring homology-directed repair (HDR), consider using an Alt-R Cas9 nickase to create single-strand breaks<\/a>. Use of a nickase variant with a pair of guide RNAs reduces off-target effects by requiring specific binding of 2 ribonucleoproteins (RNPs). However, you will need 2 potent CRISPR target sites at an appropriate distance from your HDR target site.<\/p>\n CRISPR RNPs.<\/strong> While Alt-R CRISPR guide RNAs are compatible with CRISPR proteins from any source (e.g., cells that stably express CRISPR protein, or CRISPR proteins expressed from DNA or mRNA constructs), we recommend delivery of CRISPR reagents as RNPs by lipofection (e.g., Figure 1), electroporation, or microinjection. Cas9 RNPs are compatible with all 3 methods; however, for Cas12a RNPs, we recommend electroporation and microinjection delivery methods.<\/p>\n <\/p>\n IDT scientists have developed detailed lipofection and electroporation protocols for using the Alt-R CRISPR-Cas9 System<\/a> and the Alt-R CRISPR-Cas12a System<\/a> in mammalian cells (Table 1). With help from our collaborators, we also make user-submitted methods available for genome editing in other model systems. These protocols and methods are a good starting point for protocol optimization for your studies, and often offer tips for all aspects of genome editing experiments, from growth conditions through genome editing detection.<\/p>\nAdvantages of using RNPs<\/h2>\n
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![\"Lipofection](\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/04815d15-3279-6e2e-aa53-ff00001c1b3c-d-ge17lt-crisprprotocols-f1.jpg\" "\\"D-GE17LT-CRISPRprotocols-F1\\"")
Protocols and starting points are increasingly available<\/h2>\n